Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase

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Cite This: Biochemistry XXXX, XXX, XXX −XXX

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and

Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa

Arylsulfatase

Bert van Loo,^†,§Ryan Berry,^‡Usa Boonyuen,^†,∥Mark F. Mohamed,^†Marko Golicnik,^†,⊥

Alvan C. Hengge,*^,‡and Florian Hollfelder*^,†

^†Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

^‡Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States

S

* Supporting Information

ABSTRACT: Pseudomonas aeruginosa arylsulfatase (PAS)

hydrolyzes sulfate and, promiscuously, phosphate monoesters.

Enzyme-catalyzed sulfate transfer is crucial to a wide variety of

biological processes, but detailed studies of the mechanistic

contributions to its catalysis are lacking. We present linear free

energy relationships (LFERs) and kinetic isotope eﬀects

(KIEs) of PAS and analyses of active site mutants that suggest

a key role for leaving group (LG) stabilization. In LFERs

PAS^WThas a much less negative Brønsted coeﬃcient

obs‑Enz

(β_{leaving group}

= −0.33) than the uncatalyzed reaction

obs

(β_{leaving group}= −1.81). This situation is diminished when

cationic active site groups are exchanged for alanine. The

considerable degree of bond breaking during the transition

state (TS) is evidenced by an ¹⁸O_bridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group

stabilization by active site K375, in cooperation with H211. ¹⁵N KIEs and the increased sensitivity to leaving group ability of the

H‑D

sulfatase activity in neat D₂O (Δβ_{leaving group}

= +0.06) suggest that the mechanism for S−O_bridgebond ﬁssion shifts, with

decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation.

18

O

KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a signiﬁcantly more associative

nonbridge

character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift.

This diﬀerence in enzyme-catalyzed TSs appears to be the major factor favoring speciﬁcity toward sulfate over phosphate esters

by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate

(charge).

occurrence in eukaryotes and prokaryotes, relevance for a variety

of key processes (e.g., development,⁵⁻¹¹germination,¹²

resistance against toxic defense molecules,^13,14mucin desulfa-

tation,¹⁵⁻¹⁷or degradation of mucopolysaccharides^18,19) and

the danger of various diseases as a result of their malfunction

(e.g., lysosomal disorders^18,20), their mechanism has not been

studied in the same detail as that of the related phosphatases.

The majority of sulfatases known are members of the alkaline

phosphatase (AP) superfamily. The mechanism of transition

state (TS) stabilization during enzyme-catalyzed substrate

hydrolysis of one member of this superfamily, Escherichia coli

alkaline phosphatase (EcAP), has been subject of a large number

of in-depth studies involving the experimental tools of linear free

energy relationships (LFERs), kinetic isotope eﬀects (KIEs),

mutant studies, and structural analysis by X-ray crystallog-

rylsulfatases catalyze the in vivo hydrolysis of sulfate

A_{monoesters, producing inorganic sulfate, typically remov-}

ing it from a sugar or a steroid hormone. Sulfatases are highly

proﬁcient enzymes, with catalytic proﬁciencies ((k_cat/K_M)/

k

_uncat) well above 1 × 10¹³to 1 × 10¹⁷M⁻¹for the model

substrate 4-nitrophenyl sulfate 1d (Scheme 1).¹⁻⁴Despite their

Scheme 1. General Reaction Scheme for the PAS-Catalyzed

Hydrolysis of Aryl Sulfate Monoesters 1a−1l and Aryl

Phosphate Monoesters 2b−2l

Received: September 18, 2018

Revised: January 11, 2019

A

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Article

raphy.²¹⁻³⁷In addition computational simulations have been

employed to pinpoint transition-state interactions.^{27,28,38−43}

Sulfatase members of the AP superfamily have been studied

less extensively. Catalytically important residues have been

shown to be conserved among the arylsulfatases^2,4,44−48and

mutant studies (e.g., of human arylsulfatase A,⁴⁹choline

sulfatase,⁴and the closely related phosphonate monoester

hydrolase⁵⁰) have suggested that many of these conserved

residues are indeed involved in the catalytic pathway in the

enzyme active site. LFERs and KIEs have been measured for a

number of phosphatases,^{21−24,36,51−59}but only one such study is

available for sulfatases.⁶⁰

In addition to the family relationship, members of the AP

superfamily are also typically catalytically promiscuous;⁶¹⁻⁶³

that is, they catalyze multiple, chemically distinct reactions with

large rate accelerations.^64,65Within the superfamily reciprocal

relationships of crosswise catalytic promiscuity are observed;

that is, the promiscuous activity of one member is the native

function of another, and vice versa.^64,65Given the postulated role

of promiscuity in evolution by gene duplication,^61,66functional

crossover deﬁnes such functional relationships as pathways for

respecialization or repurposing in enzyme superfamilies,⁶⁷

which was experimentally demonstrated recently by a more

than 10⁷-fold speciﬁcity switch of a sulfatase to enhance

phosphonate hydrolase activity.⁶⁸As catalysis for the activity

under selective pressure must be maintained at a relevant level

during evolution, the question of how mechanistic features of

these enzymes can be eﬀective for catalysis of diﬀerent reactions

arises.

In this work we studied the reactions catalyzed by the

promiscuous arylsulfatase from Pseudomonas aeruginosa (PAS)

using LFERs and KIEs. PAS has a wide active site opening⁴⁴

andin contrast to sulfatases with high speciﬁcity for a

particular leaving group (such as a sugar moiety^69,70or

choline⁴)accepts a range of aromatic substrates: so that the

construction of LFERs based on series of aryl sulfates of diﬀerent

reactivity is possible with minimal interference from unique

binding eﬀects. PAS operates at high rates (k_cat/K_M= 4.9 × 10⁷

s⁻¹M⁻¹), even for the hydrolysis of the non-natural substrate 4-

nitrophenyl sulfate 1d.¹In addition, PAS promiscuously

catalyzes the hydrolyses of phosphate mono-¹and diesters⁶³

as well as phosphonates,⁷¹thus covering mechanistically distinct

hydrolase reactions.⁷²However, despite its catalytic promiscu-

ity, PAS is a genuine sulfatase: it is typically expressed under

sulfate starvation conditions, part of an operon coding for

sulfate-processing enzymes, and thought to act as a sulfate

scavenger.^73,74

Figure 1. Active site and reaction mechanism of PAS. (a) 3D

representation of the active site of PAS with a bound sulfate ion.⁴⁴The

assignment of the analogous nonbridging (O₁−O₃) and bridging (O₄)

oxygens in the sulfate ester substrate were based on positions of the

active site residues in the X-ray structure of human arylsulfatase A

C69A⁷⁵with p-nitrocatechol sulfate bound in the active site. (b)

Proposed catalytic pathway for PAS-catalyzed sulfate monoester

hydrolysis. When the sulfate monoester substrate is bound, the

hydrated fGly51 performs a nucleophilic attack on the sulfur atom, and

the bond to the alcohol leaving group (ROH) is broken (S−O_bridge

bond ﬁssion) (step 1). The covalent intermediate is broken down by

base-catalyzed hemiacetal cleavage in which inorganic sulfate acts as the

leaving group (step 2). The enzyme is subsequently regenerated by

hydration of the formylglycine aldehyde (step 3). (c) Schematic

representation of the steps in (b). k₁is the rate of formation of the

enzyme−substrate (ES) complex from free enzyme (E) and substrate

(S), and k₋₁represents the dissociation rate of the ES complex. k₂is the

rate constant for the nucleophilic attack of the hydrated formylglycine

and subsequent S−O_bridgebond ﬁssion (step 1), and k₃is that of

hemiacetal cleavage (step 2). The rehydration of the fGly residue (step

3) is expected to be several orders faster that hemiacetal cleavage and

thus was not considered for the interpretation of pre-steady-state

kinetics. Product P₁is the phenolate leaving group expelled from the

substrate in step 1; product P₂is inorganic sulfate.

On the basis of the X-ray structure of PAS⁴⁴(and those of the

human arylsulfatases A^47,75(hASA) and B⁴⁵(hASB) combined

with mutant data for hASA⁴⁹) a double displacement catalytic

pathway was proposed, in which a post-translationally modiﬁed

cysteine,⁷⁶formylglycine fGly51, performs a nucleophilic attack

on the sulfur center (Figure 1b). The covalent hemiacetal

intermediate is broken down with assistance of H115 acting as a

general base (step 2 in Figure 1b). Finally the aldehyde form of

the fGly nucleophile is again hydrated, regenerating the enzyme

for the next round of catalysis. Mutant data for several of the

analogous active site residues in hASA show that a single

mutation of one of the residues likely to be involved in charge

compensation during the TS results in lowered but still

detectable levels of activity.⁴⁹During the hydrolysis of sulfates,

negative charge is expected to build up in the TS on the sulfuryl

group and on the leaving group.^3,77TS stabilization can be

achieved by oﬀsetting this charge build-up: using positively

charged functionalities such as metal ions or by using hydrogen

bonding or electrostatic interaction with positively charged

amino acid side chains. The latter possibly may involve proton

transfer to the leaving-group oxygen. The active site of PAS

contains a number of residues that could neutralize charge build-

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up on oxygen atoms during the transition state or aﬀect the pK_a

of the formylglycine (fGly51) nucleophile (Figure 1).

loaded onto a Superdex 200 prep grade gel ﬁltration column.

PAS eluted at the expected elution volume of monomeric PAS.

Protein-containing fractions were pooled and concentrated to

100−350 μM in 50 mM Tris-HCl and aliquoted in appropriate

portions, ﬂash frozen in liquid N₂, and stored at −20 °C. Protein

concentrations were calculated based on the molar extinction

coeﬃcient at λ = 280 nm, ε₂₈₀= 102 790 M⁻¹cm⁻¹, calculated

from the amino acid sequences using ProtParam (http://expasy.

org/tools/protparam.html).

Construction of Mutants. All mutants of PAS, except for

mutant C51S, which was constructed previously,¹were made by

site-directed mutagenesis according to the QuikChange

protocol (Agilent), using primers listed in Table S16,

Supporting Information and the appropriate template plasmid.

Enzyme Kinetics. All data for steady-state enzyme kinetics

were recorded at 25 °C in 100 mM Tris-HCl pH 8.0

supplemented with 500 mM NaCl or as indicated. Observed

initial rates (V_obs) were determined by following an increase in

absorbance at a ﬁxed wavelength (ranging from 270 to 400 nm

depending on the substrate) as a result of product formation

over time in microtiterplates (Spectramax Plus, Molecular

Devices) or quartz cuvettes (Varian 100 Bio). Catalytic

parameters k_cat, K_M, and/or k_cat/K_Mwere obtained by ﬁtting

the dependency of V_obson substrate concentration ([S]) at a

ﬁxed enzyme concentration ([Enz]) (eq 1).

Here we provide a detailed quantitative examination of the

interaction of active site residues with the TS in the arylsulfatase-

catalyzed reaction. We use LFER and KIE data to compare the

nature of the TS for the native PAS-catalyzed sulfate monoester

hydrolysis with the uncatalyzed reaction and show that, while

both are dissociative, the TS of the enzyme-catalyzed reaction

has somewhat more associative character. The latter diﬀerence is

absent for phosphate monoester hydrolysis. Finally, changes in

LFERs and KIEs as a result of alanine scanning mutagenesis of

PAS active site residues suggest that K375 serves as the general

acid that minimizes leaving group charge change in the TS and

provides leaving group stabilization.

MATERIALS AND METHODS

■

Sulfate Monoester Compounds for Linear Free Energy

Relationships and Kinetic Isotope Eﬀect Studies. Sulfate

monoester 1d and phosphate monoesters 2d and 2k (Scheme 1)

were purchased from Sigma. Sulfate esters were synthesized

from the respective phenol and chlorosulfonic acid; phosphate

monoesters were synthesized from phosphoryl chloride and the

respective phenol. Detailed procedures are given in the

Supporting Information. The isotopically labeled forms of 4-

nitrophenyl sulfate (1d)⁷⁸and phosphate (2d)⁵³for measure-

ment of kinetic isotope eﬀects were synthesized as described

previously.

k_cat× [Enz] × [S]

V

=

obs

K_M+ [S]

(1)

Protein Production and Puriﬁcation. Expression of

recombinant Pseudomonas aeruginosa Arylsulfatase (PAS,

Uniprot ID K9NCQ5) from plasmid pME4322⁷⁹and derived

mutants was done in E. coli BL21 (DE3) growing in lysogeny

broth (LB) or yeast−trypton medium (2YT) containing 30 mg

mL⁻¹kanamycin. The cells were grown at 37 °C, until an OD₆₀₀

of ∼0.6−0.8 was reached. The culture was cooled to 30 (WT) or

20 °C (mutants), isopropyl β-D-1-thiogalactopyranoside

(IPTG) was added up to 0.75 mM, and the culture was grown

for 4 h at 30 °C (WT) or overnight at 20 °C (mutants).

Cells expressing PAS were harvested by centrifugation and

resuspended in 50 mM tris(hydroxymethyl)aminomethane

(Tris) HCl pH 8.0. One tablet of complete ethylenediaminete-

traacetic acid (EDTA)-free protease inhibitor cocktail (Roche)

per 12 g of wet cell pellet was added to the suspension, and the

cells were lysed either by using an emulsiﬂex-C5 homogenizer

(Avestin) or by sonication. Cell-free extract (CFE) was obtained

by centrifugation of the crude cell lysate at 30 000g for 90 min.

The PAS variants were puriﬁed from CFE by subsequent anion

exchange (Q-sepharose), hydrophobic interaction (phenyl

sepharose), and size exclusion (Superdex 200) chromatography.

All steps were performed in 50 mM Tris-HCl pH 8.0 with the

appropriate additive for each step. The anion exchange

chromatography was performed as described before.¹Protein-

containing fractions that eluted from the anion exchange column

were pooled, and the combined fractions were brought to 200

mM (NH₄)₂SO₄by adding the appropriate volume of 50 mM

Tris-HCl pH 8.0 + 2 M (NH₄)₂SO₄and subsequently loaded

onto a phenyl sepharose hydrophobic interaction column. The

column was washed with two column volumes (CV) of 50 mM

Tris-HCl pH 8.0 + 200 mM (NH₄)₂SO₄. Protein was eluted

from the column with a gradient of 200−0 mM (NH₄)₂SO₄in

50 mM Tris-HCl over ﬁve CV followed by a further ﬁve CV with

50 mM Tris-HCl pH 8.0. Fractions containing active protein

were pooled and concentrated into 50 mM Tris-HCl pH 8.0 to

∼10−15 mg mL⁻¹protein. The concentrated protein was

The dependency of the various kinetic parameters (k_cat, 1/K_M,

and k_cat/K_M, represented by K in eq 2) on leaving group ability

(as represented by their pK_avalue) was ﬁtted to eq 2 to obtain

the observed Brønsted constants for leaving group dependence

(β_{leaving group}^obs).

obs

leavinggroup

log[K] = β

× pK_a^leavinggroup+ C

(2)

Stopped-Flow Kinetics. Fast kinetics for PAS WT-

catalyzed hydrolysis of sulfate monoester 1d were recorded

using a SX20 stopped-ﬂow setup (Applied Photophysics). No

unambiguous burst phase could be detected with PAS

concentrations between 1 and 8 μM and 1 mM sulfate

monoester 1d, with 100 mM Tris-HCl pH 8.0, containing 500

mM NaCl at 20 °C. Data are shown in Figure S3.

Kinetic Isotope Eﬀects (KIEs). Natural abundance 1d or 2d

was used for measurements of ¹⁵(V/K). The ¹⁸O KIEs ¹⁸(V/

K)_bridgeand ¹⁸(V/K)_nonbridgewere measured by the remote label

method, using the nitrogen atom in p-nitrophenol as a reporter

for isotopic fractionation in labeled bridging or nonbridging

oxygen positions.⁸⁰The particular isotopic isomers used are

shown in the Supporting Information. Isotope eﬀect experi-

ments used 100 μmol of substrate, at 25 °C in 50 mM Tris

buﬀer, pH 8.0. The substrate concentration was 19 mM, and the

reactions were started by addition of wild-type or mutant

enzyme, 1 μM for substrate 1d, and 725 μM for substrate 2d.

After reactions reached completion levels between 40% and 60%

they were stopped by titration to pH 3 with HCl. Protocols for

isolation of p-nitrophenol, isotopic analysis, and calculation of

the isotope eﬀects were the same as previously described,^53,78

and they are described in the Supporting Information.

RESULTS AND DISCUSSION

■

Determination and Interpretation of Steady-State

Parameters. Puriﬁed PAS WT¹was used to determine

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Figure 2. Dependence of catalytic parameters k_cat(blue) 1/K_M(red) and k_cat/K_M(black) (log values) for PAS-catalyzed hydrolysis of sulfate

monoesters on leaving group ability (as represented by the pK_aof the free phenol in solution). (a) PAS WT (see for a superposition of the data

measured here with that of Williams et al.⁶⁰in Figure S6, Supporting Information); (b) PAS C51S; (c) PAS K113L; (d) PAS H115A; (e) PAS H211A;

(f) PAS K375A; (g) PAS C51S/K375A; (h) PAS H115A/K375A; (i) PAS WT with phosphate monoesters. All data were obtained in 100 mM Tris-

HCl pH 8.0; 500 mM NaCl at 25 °C. The resulting slopes (=β_{leaving group}^obs) are listed in Tables 1 and S14. The data for the unsubstituted phenol sulfate

monoester 1k deviated signiﬁcantly from the trend for all enzymes, suggesting a consistent unique diﬀerence in binding compared to the other sulfate

monoester substrates and were therefore not included in any of the ﬁts. Data points represented by open circles were not included in the ﬁts: e.g., 1f in

panel (c), in which the ﬁtted value for K_Mdeviated by an order of magnitude; 1a−1d in panel (f) for the ﬁt in blue, where the slope for k_catwas only

expected to be relevant for the TS if fully representing the rate-limiting step: this is only the case for leaving groups with pK_a> 8, so other points were

excluded to reﬂect the chemical reaction in β_{leaving group}^obsas much as possible; see also Figure S10, Supporting Information; 1e in panel (g) and 1b in

panel (h) were excluded, because one parameter deviated strongly, again suggesting idiosyncratic eﬀects in these substrates. All data for the kinetic

parameters are listed in the Supporting Information (Tables S1 and S3 −S10).

Michaelis−Menten parameters k_cat, K_M, and k_cat/K_Mfor a series

of phenyl sulfate monoesters (compounds 1a−1l, Scheme 1)

with varying leaving group abilities (represented by their pK_a

values, which range from 5.2 to 10.35 (Table S1 and Figures S1

and S2, Supporting Information). These substrates provide

insight into the nature of the TS of PAS-catalyzed sulfate

monoester hydrolysis by allowing the construction of LFERs,

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similar to those reported for several phosphoryl transfer

enzymes.^{22,23,32,33,56−59,72,81,105}

comparison of both studies (see Figure S6, Supporting

Information) shows that most points superimpose well with

our data but that the following factors seem to have resulted in a

distortion of the slope due to idiosyncratic eﬀects of substrates

(e.g., due to steric clashes or interactions between active site

residues and phenolate substituents): (i) the choice of leaving

groups with higher pK_a(speciﬁcally the inclusion of the bulky 4-

amino-acetyl- and 4-methoxyphenolate), (ii) the narrower range

of pK_avalues (three compared to almost ﬁve log units covered

here) and (iii) basing the study on overall fewer data points (7 vs

12 in our work) with worse signiﬁcance (p-value, 0.026 vs less

than 1 × 10⁻⁴) and correlation coeﬃcients (R², 0.66 vs 0.91).

The underlying meaning of the steady-state parameters used

to monitor the bond-making and -breaking processes must be

carefully considered. Since the steady-state catalytic rate

constant (k_cat) was shown to be essentially independent of

leaving group ability (Figure 2a, varying between 9 and 18 s⁻¹,

Table S1, Supporting Information), it does not reﬂect the

substrate reactivity. On the basis of the kinetic scheme for

enzyme-catalyzed substrate hydrolysis (Figure 1c), the relevant

rate constant to probe the nature of the TS should be k₂. If

enzyme-catalyzed cleavage of the sulfate ester bond were fully

rate-limiting, the steady-state catalytic rate constant k_catwould

be essentially equal to k₂(equation S14, Supporting

Information). If this is not the case, that is, if k₂≥ k₃, more

complex terms arise (see equations S1 −3, Supporting

Information). Assuming that the substrate binding constants

(K_D= k₋₁/k₁) are similar for the complete series of sulfate

monoesters (Scheme 1), k_cat/K_Mreports on all steps from free

enzyme and substrate to the ﬁrst irreversible transition state. In

this case the formation of the covalent intermediate between the

formylglycine nucleophile and the respective sulfur center of the

substrates that leads to leaving group departure is likely to be

irreversible, as no signiﬁcant inhibition by the phenolate product

is observed (see equations S1 −S3 in the Supporting Information

for details on the relation between the Michaelis−Menten

parameters and the individual rate constants for the various

enzymatic steps).

obs

Starting from the quantitatively diﬀerent values for β_{leaving group}

our analysis below naturally diﬀers from that of Williams et al.

However, the slope β_{leaving group}^obs, is less steep compared to that

for the solution reaction in both studies, despite the quantitative

diﬀerences.

A possible cause for the considerably less negative

obs

β_{leaving group}compared to the solution reaction could be that

the k_cat/K_Mvalues do not only represent a chemical step, as

shown previously for wild-type E. coli alkaline phosphatase

(EcAP).^35,53,83However, considerations summarized in the

Supporting Information (section H) rule out diﬀusion control

obs

and suggest that the deviation of β_{leaving group}

from the

β_{leaving group}of the solution reaction is a genuine eﬀect of

substrate binding and turnover by the enzyme.

Previous experimental studies into the nature of the TS of

enzyme-catalyzed sulfate transfer using LFERs^60,84typically

showed less steep correlations than the corresponding solution

reaction,⁸⁵⁻⁸⁸bringing the Brønsted slopes to values closer to

zero. This decrease could be ascribed to interactions with

cationic groups in the active site, but no KIEs or mutational data

were available to check this hypothesis. In addition an LFER for

sulfate transfer has been reported for the promiscuous sulfatase

activity of AP WT.³¹Subsequent KIE studies showed that in this

case a dissociative TS is likely.²¹In AP the less negative

β_{leaving group}^obscompared to the solution reaction could arise from

interaction of the leaving group with positively charged moieties

in the enzyme active site, most likely a divalent metal ion (Zn²⁺).

The latter phenomenon has also been reported for AP-catalyzed

phosphate monoester hydrolysis.⁸⁹In several protein tyrosine

phosphatases a protonated aspartate was identiﬁed as

responsible for leaving group stabilization.^{51,52,54−56,59}Replace-

ment of this aspartate with asparagine restored the leaving group

dependence to a value close to that of the solution reaction.⁵⁶In

protein phosphatase 1 (PP1), a histidine was proposed to

assume the same role, although practical limitations (low yield

and poor activity) prevented experimental veriﬁcation.⁵⁸

LFER for PAS WT-Catalyzed Phosphate Monoester

Hydrolysis. The fact that PAS WT is also a proﬁcient

phosphatase¹((k_cat/K_M)/k_uncat= 2.9 × 10¹¹M⁻¹toward

phosphate monoester 2d) opens the possibility of studying

two reactions that proceed through similar TSs in solu-

tion^{31,53,77,82,90,91}in a single active site and also facilitates

comparisons with the more widely studied phosphatases.

Michaelis−Menten parameters were determined for a series

of phosphate monoesters (2b−2l, Scheme 1). As for the

sulfatase reaction, k_catis practically independent of the leaving

group pK_a(varying between 0.6 and 1.2 × 10⁻²s⁻¹, Figure 2i and

Table S3, Supporting Information), suggesting that the rate-

limiting step for phosphate as well as sulfate monoesters is not

leaving group-dependent. The K_Mvalues increase with leaving

group pK_aand range from 0.03 to 0.92 mM, ca. 100-fold higher

LFER for PAS WT-Catalyzed Sulfate Monoester

Hydrolysis. Plots of k_cat/K_Magainst the leaving group pK_a

(Figure 2) were linear, with a β_{leaving group}^obsof −0.33 (Table 1)

that was considerably less negative than that of the rate for the

uncatalyzed hydrolysis reaction (k_uncat), for which a

β_{leaving group}^obsof −1.81 has been measured.³This work partially

reproduces a study of Williams et al.,⁶⁰who recently constructed

an LFER for PAS but arrived at a much steeper slope (−0.86). A

Table 1. Overview of the Observed Brønsted Constants for

Leaving Group Dependence (β_{leaving group}^obs) of k_cat/K_Mfor

PAS-Catalyzed Sulfate and Phosphate Monoester

a

Hydrolysis

obs

reaction

catalyst

β_{leaving group}

b

sulfate monoesters

solution (neutral)

solution (monoanion)

WT

C51S

K113L

H115A

H211A

K375A

C51S/K375A

K113L/K375A

H115A/K375A

H211A/K375A

solution (monoanion)

solution (dianion)

WT

−0.27

−1.81

b

−0.33 0.04

−0.43 0.05

−0.64 0.06

−0.67 0.05

−0.55 0.07

−0.94 0.04

−0.76 0.24

c

nd

−1.04 0.06

c

nd

d

phosphate monoesters

−0.27

−1.23

d

−0.39 0.04

a

All enzymatic reactions performed in 100 mM Tris-HCl pH 8.0, 500

mM NaCl, 25 °C. Edwards et al.,³H₂O as the nucleophile. Not

b

c

determined. Activity too low to be detectable. Kirby & Varvoglis,⁸²

d

H₂O as the nucleophile.

E

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than for sulfatase activity. The slope of the Brønsted plot

β_{leaving group}^obsfor k_cat/K_Mfor phosphate monoester hydrolysis is

−0.39 0.04 (Table 1), identical within the error margins to the

value observed for the sulfatase reaction (and conﬁrmed by a

cross-correlation graph with a slope of unity; see Figure S9,

Supporting Information). As observed for the sulfatase reaction,

enzyme-catalyzed phosphate monoester hydrolysis is consid-

erably less sensitive to leaving group ability than the solution

data for the enzyme−substrate complex of an inactive variant of

human arylsulfatase A.⁷⁵The majority of the interactions with

the nonbridging and leaving group oxygens are expected to be

provided by amino acid side chains, which opens the possibility

of assessing the importance of the correction factors of eq 3

experimentally by determining the leaving group dependence of

active site mutants of PAS. To this end the nucleophile (fGly51)

and a residue that directly interacts with it (H115) were mutated

to assess the contribution of the nucleophile. Furthermore,

several positively charged groups expected to provide charge

compensation during the GS and TS by interacting with

nonbridging (K113 and K375) and leaving group (H211 and

K375) oxygens were removed by mutating the respective

residues into alanine or leucine.

reaction for the phosphate monoester dianion (β_{leaving group}

=

−1.23⁸²). As for sulfate monoester hydrolysis, these consid-

erable deviations can be caused by stabilization/masking of

locally developing negative charge, in particular, on the leaving

group oxygen. This idea is probed in the next paragraph. The less

negative value for β_{leaving group}is also consistent with an

intrinsically more associative nature of the respective enzymatic

TSs (compared to the solution reaction), possibly reinforced by

the presence of cationic groups in the active site. This

interpretation assumes the same degree of nucleophilic

involvement. If this is not the case, the β_{leaving group}value will

only reﬂect the extent of leaving group cleavage. However,

kinetic isotope eﬀect studies (see below for details) discounted

these possibilities and suggested that the TS is similar (but not

identical) to the solution reaction, as in previously studied

All mutants were puriﬁed to homogeneity and Michaelis−

Menten parameters determined for the same series of sulfate

monoesters (1a−1l, Scheme 1) as used with the wild-type

enzyme. The mutations resulted in 1 × 10³to 1 × 10⁸-fold drops

in catalytic eﬃciencies (k_cat/K_M) for the various substrates

(Tables S4 −S8, Supporting Information). For mutants C51S

and H211A k_catwas still independent of leaving group ability

(Figure 2b,e, respectively), suggesting that, as in the wild-type

enzyme, the leaving group-dependent step is not rate limiting.

However, the k_catfor these mutants was reduced ∼1 × 10³-fold

(C51S) and ∼1 × 10⁵-fold (H211A) compared to PAS WT. The

K_Mvalues for PAS C51S and H211A were within an order of

magnitude of those for the wild-type enzyme (Tables S4 and S7)

and, as observed for wild-type PAS, the β_{leaving group}^obsvalues for

k_cat/K_Mand 1/K_Mare nearly identical. For PAS K113L and

H115A both k_catand 1/K_Mdecreased with increasing leaving

group ability (Figure 2c,d, Tables S5 and S6), albeit it only at the

higher end of the pK_aspectrum for H115A. For PAS K375A k_cat

decreases with increasing leaving group pK_a, indicating that the

rate-limiting step is largely leaving group-dependent. The K_M

values are increased ∼1 × 10³-fold compared to the wild-type

enzyme and are largely constant (varying in a range of 5−10

mM) for the substrates with a leaving group pK_a> 8 (Figure 2f,

phosphatases.^{23,37,56,58,59}

The Eﬀect of Mutations on the β_{leaving group}

obs

for

Sulfate Monoester Hydrolysis. As discussed above, the

β_{leaving group}^obsof enzyme-catalyzed phosphate and sulfate transfer

reactions can be inﬂuenced by compensation by positively

charged moieties in the active site of the negative charge build-

up that occurs during the TS.^{23,33,37,58,84}Furthermore the

change in nucleophile between the solution (H₂O) and enzyme-

catalyzed (formylglycine) reaction can also inﬂuence the

β_{leaving group}^obs. Zalatan et al.³⁷formulated these considerations

to be able to calculate expected diﬀerences in leaving group

dependence between enzyme-catalyzed (β_{leaving group}^Enz) and

solution reactions (β_{leaving group}^solution) (eq 3). The expected

change in leaving group dependence resulting from a change in

nucleophile between the enzyme-catalyzed (fGly) and solution

(H₂O) reaction is calculated from the diﬀerence in nucleophil-

icity (pK_nuc^Enz− pK_nuc^solution) weighed with the sensitivity of the

leaving group dependence of the reaction type to a change in

nucleophile (p_xy). The second of the contributing factors is

leaving group-dependent binding of the ground state (GS,

β_bind^GS) and TS (β_bind^TS). For k_cat/K_M-based β_{leaving group}values

the latter two are indistinguishable from each other and are

treated as a single variable (∑β_bind= β_bind^GS+ β_bind^TS).

obs

Table S8). The β_{leaving group}for k_cat/K_Mis nearly identical to

that for k_catfor pK_a> 8. As for PAS WT, the LFERs for k_catand 1/

K_Mfor PAS K113L, H115A, and K375A could be simulated

based on assumed values for the pre-steady-state kinetic

parameters (Figure S10, Supporting Information). In particular,

for K113L and K375A the break in the LFER for k_catcould be

explained by a change of the rate-limiting step with increasing

pK_a^{leaving group}

.

obs

The β

of all active-site mutants was less negative than

leaving group

obs

obs‑WT

the β

for the wild type. Δβ_{leaving group}(=β

−

leaving group

Enz

leaving group

solution

leaving group

Enz

nuc

solution

)

nuc

obs mutant

β

) was calculated as the slope of the linear correlation

β

= β

+ p × (pK

− pK

leaving group

xy

of log[(k_cat/K_M)_WT/(k_cat/K_M)_mutant] versus pK_a^{leaving group}(Table

2, Figure S11a). As described above the diﬀerence in leaving

group dependencies between enzyme-catalyzed and uncatalyzed

sulfate and phosphate monoester hydrolysis is inﬂuenced by the

nature of the nucleophile and charge compensation eﬀects (eq

3). On the basis of the pH-rate proﬁle for PAS-catalyzed sulfate

monoester hydrolysis the pK_nucof the enzyme is expected to be

less than 7.2.¹The p_xyvalue for sulfate transfer is not known, but

it is expected to be similar to the value for phosphate monoesters

TS

bind

GS

bind

+ β

(3)

For E. coli AP, in particular, the interaction of the leaving

group oxygen with one of the two Zn²⁺ions during the GS and

TS was thought to be mainly responsible for the ∑β_bindof

+0.33⁸⁹(3 times larger than the expected contribution of the

change in nucleophile from H₂O to the active site serine of

+0.11). Selective removal of only the Zn²⁺ion responsible for

leaving group stabilization is not feasible, and therefore

experimental assessment of these calculations was not possible.

The assignment of the analogous positions of the nonbridging

and bridging (leaving group) oxygens in an inorganic sulfate

molecule with the active site residues of PAS (as shown in the X-

ray structure,⁴⁴Figure 1) was based on homology with structural

(0.013).⁹²Assuming that the pK_nuc≈ 6 and pK_nuc

=

Enz

solution

−1.7 (nucleophile = H₂O), the eﬀect of the change in

nucleophile between the PAS WT-catalyzed and the solution

reaction is expected to be 0.013 × (6 − (−1.7)) = +0.10.

Assuming the diﬀerence in nucleophilicity between fGly

(solution pK_a≈ 13−14⁹³) and serine (pK_a≈ 16, similar to

F

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Biochemistry

Article

detection limit). This observation strengthens the idea that the

eﬀect of each mutation is buﬀered by the nearby presence of a

residue that can take over its function: if the measured

reductions in activity for the single mutants were simply

additive, the expected k_cat/K_Mfor sulfate monoester 1d should

be 2.6 × 10⁻³s⁻¹M⁻¹(according to equation S21, Supporting

Information) and still be detectable. By contrast, their

cooperativity leads to a larger detrimental eﬀect, when both

are removed.

Table 2. Eﬀect of Mutations on Leaving Group Dependence

for PAS-Catalyzed Sulfate Monoester Hydrolysis in PAS WT

and K375A

obs

mutation

C51S

reaction

eﬀect in

Δβ_{leaving group}

a

b

a

b

a

1a−1l

1a−1d

1c−1l

1a, 1c−1l

1a−1l

1c−1l

WT

K375A

WT

K375A

WT

+0.10 0.04

+0.01 0.33

+0.24 0.05

+0.32 0.03

+0.15 0.05

+0.16 0.07

+0.61 0.03

K113L

H115A

As discussed above the eﬀect of leaving group-dependent

ground- and transition-state binding (∑β_bind) is expected to be

modest for residues that interact mainly with the nonbridging

oxygens: ∼10% (for all the nonbridging oxygens combined) of

the value expected for leaving group oxygen.⁸⁹This consid-

eration suggests that the maximal combined eﬀect of K113,

K375, and Ca²⁺on ∑β_bindvia interactions with the nonbridging

H211A

K375A

1a−1e

WT

a

Determined as the slope for log[(k_cat/K_M)_WT/(k_cat/K_M)_mutant

]

b

plotted versus leaving group pK_a(Figure S10a). Determined as the

slope for log[(k_cat/K_M)_K375A/(k_cat/K_M)_{double mutant}] plotted versus

leaving group pK_a(Figure S10b, Supporting Information).

obs‑WT

oxygens is expected to be ca. +0.13 (10/110 × (β_{leaving group}

− β_{leaving group}^{obs‑solution}) = 0.09 × (−0.33 − (−1.81)) = +0.13).

Since removal of a positive charge is expected to increase the

pK_nucof the enzymatic reaction, the observed eﬀect on ∑β_bindof

the interaction with nonbridging oxygens, as a result of removing

any of these three functional groups, is expected to be less than

that of ethanol) in solution translates into the same diﬀerence in

the enzyme active site, a small increase in the contribution of the

nucleophile term in eq 3 is expected for mutant C51S. On the

WT‑C51S

basis of this assumption the Δβ_{leaving group}

is expected to

be small and negative. However, we measure a value of +0.10

(Table 2). Since fGly is interacting directly with the Ca²⁺ion,

changing it to a serine may cause a change in the charge

+0.05. However, mutation K113L results in a

WT‑K113L

Δβ_{leaving group}

of +0.24. This large eﬀect partly explains

compensation eﬀects provided by the divalent cation, which

why removal of H115 has an unexpectedly large

WT‑C51S

WT‑H115A

could explain the positive value for Δβ_{leaving group}

.

Δβ_{leaving group}

of +0.32, since H115 and K113 interact

Removal of H115 is expected to result in a slight increase in

the pK_aof the nucleophile, again predicting a small negative

Δβ_{leaving group}^WT‑H115Aas a result. However, the measured value of

+0.32 suggests that any small eﬀect of the mutation on the

nucleophile term is overshadowed by a considerable decrease in

closely in the three-dimensional structure of PAS. The large

eﬀect of mutations C51S, K113L, and H115A on PAS activity

suggests that these mutations inﬂuence the interaction of the

H211A/K375A pair with the leaving group oxygen. If the eﬀects

were electrostatic and isolated from the interactions with the

leaving group oxygens, the eﬀect of these mutations would be

expected to be identical in K375A and WT; that is, these three

mutations should be additive to mutation K375A. The

combined eﬀects of K113L and K375A result in completely

inactive enzyme (k_cat/K_M< 5 × 10⁻⁶s⁻¹M⁻¹for sulfate

monoester 1d), which is lower than the expected value (7.0 ×

10⁻⁴s⁻¹M⁻¹; based on eq S21, Supporting Information) for an

additive eﬀect of both mutations. This can be explained by

interaction of both K113 and K375 with the nonbridging

oxygens, in which case their simultaneous removal most likely

results in complete abolition of substrate binding. The

introduction of mutations C51S and H115A into the K375A

variant does result in enzymes with detectable activities.

∑β_bind

.

The removal of the residues that directly interact with the

leaving group oxygen is expected to have a large eﬀect on ∑β_bind

,

whereas groups that interact only with the nonbridging oxygens

are expected to contribute to ∑β_bindat ∼10% of the value

expected for direct interactions with the leaving group oxygen.³⁷

WT‑H211A

Mutation of H211 resulted in a Δβ_{leaving group}

of only

+0.16, even though this residue interacts exclusively with be the

leaving group oxygen. Removal of the nearby K375 results in a

WT‑K375A

Δβ_{leaving group}

of +0.61. Since the removal of a positive

charge is expected to increase the pK_nuc^Enz, the actual eﬀects on

∑β_bindmay be slightly higher than observed.

Taken together these data suggest that K375 is largely

responsible for leaving group stabilization, either by electrostatic

charge oﬀset or by proton transfer. However, unlike in

PTPase,⁵⁶where a protonated aspartate performs the same

function, its removal does not result in a near-complete abolition

obs

However, the eﬀect on the β_{leaving group}is much lower in the

K375A mutant than in the wild-type enzyme, if present at all

(C51S in K375A has no signiﬁcant eﬀect on β_{leaving group}; see

Table 2 and Figure S10b for details). The nonadditive behavior

obs‑Enz

obs‑solution

of the diﬀerence between β

and β

, since

leaving group

PAS K375A‑solution

suggests that the unexpectedly large value of

Δβ

= +0.87. Values of ca. +0.1−0.2 would be

expected in case of complete removal of charge compensation

leaving group

WT mutant

Δβ_{leaving group}

for both these mutations is due to their

eﬀect on the interactions between the leaving group oxygen and

K375; that is, this eﬀect cannot be achieved unless K375 is

present. Possible explanations for this phenomenon are (i)

changes in the overall electrostatic character of the active site as

result of the mutations that decrease the strength of the

interaction between K375 and the leaving group oxygen or (ii)

changes in substrate positioning that cause the optimal

conﬁguration of the K375 leaving group oxygen paring to be

distorted, resulting in a lower contribution of this interaction to

TS stabilization.

on the leaving group as a result of the mutation (i.e., ∑β_bind= 0,

only the change in nucleophile results in a small positive

obs

Δβ

). This observation could be rationalized by partial

leaving group

compensation of the loss of charge oﬀset (or of partial proton

transfer) provided by K375 by nearby H211. The same

phenomenon (i.e., that K375 partially assumes the role of

H211) could explain the relatively small eﬀect of mutation

obs

leaving group

H211A on β

. The combined eﬀects of these two

residues ﬁnd support in the double mutant enzyme PAS

H211A/K375A, for which no sulfatase activity was detectable

(k_cat/K_M< 5 × 10⁻⁶s⁻¹M⁻¹for sulfate monoester 1d; see

Supporting Information for consideration of the experimental

In addition (or as an alternative) to the above considerations

of charge compensation, changes in β_{leaving group}^obscan also reﬂect

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changes in the nature of the transition state. We discuss these

aspects together with kinetic isotope eﬀects below.

Kinetic Isotope Eﬀects. Kinetic isotope eﬀects were

measured for the bridging (or leaving group) and nonbridging

oxygens as well as the nitro group for enzyme-catalyzed

hydrolysis of sulfate monoester 1d and phosphate monoester

2d (Figure 3; Chart S1, Supporting Information), to comple-

The expected ranges of the isotope eﬀects in sulfate

monoester 1d and phosphate monoester 2d and their

interpretation have been discussed in detail previously.^80,95

The secondary KIE at the nitrogen atom, ¹⁵(V/K), reports on

negative charge development on the nitrophenolate leaving

group in the transition state. The p-nitrophenolate anion has

contributions from a quinonoid resonance form, with decreased

N−O bond order and increased N−C bond order. Because N−

O bonds have tighter vibrational frequencies, the nitrogen atom

is more tightly bonded in neutral p-nitrophenol (or in substrates

1d and 2d) than in the phenolate anion.⁹⁶Thus, the ¹⁵N KIE for

deprotonation of p-nitrophenol is normal. When protonation or

other interactions maintain the leaving group in a neutral state,

there is no isotope eﬀect (KIE = unity). This KIE reaches its

maximum value of ∼1.003, reﬂecting a full negative charge,

when the leaving group in the TS has a very high degree of bond

ﬁssion and no interactions neutralize the resulting charge. The

KIE at the bridging oxygen atom, ¹⁸(V/K)_bridge, is a primary

isotope eﬀect that arises from S−O or P−O bond ﬁssion and is

also aﬀected by O−H bond formation, if the leaving group is

simultaneously protonated in the TS. Bond ﬁssion produces

normal isotope eﬀects, primarily due to reduction of the

stretching vibration in the TS as the force constant is lowered.

Protonation of this atom in the TS gives rise to inverse eﬀects,

from the new vibrational modes introduced from the forming

bond. A large body of data from phosphate and sulfate ester

hydrolysis shows the isotope eﬀect from P−O or S−O bond

ﬁssion is normally larger in magnitude than the inverse eﬀect

from protonation. A normal ¹⁸(V/K)_bridgeeﬀect near its

maximum of 1.03 reﬂects a largely broken S−O or P−O bond

in the TS, arising from loss of vibrations involving this bond. In

native enzymes utilizing general acid catalysis, or uncatalyzed

reactions under acidic conditions, the observed ¹⁸(V/K)_bridgeis

reduced by protonation, as shown in Table 3.

Figure 3. Positions for the KIE measurements in sulfate monoester 1d

and phosphate monoester 2d. Eﬀects were measured for the

18

nonbridging oxygens (a,

O

KIE), bridging oxygen (b, ¹⁸O_bridge

nonbridge

KIE), and for the nitro group (c, ¹⁵N KIE). See Chart S1 (Supporting

Information) for the structures of the labeled compounds used.

ment the Brønsted analysis above.^56,58The KIE experiment

requires turnover of approximately half of a 100 μmol sample of

labeled substrate. With mutants of low activity this can require

long reaction times, or unpractically large amounts of enzyme.

For that reason, most but not all of the mutants for which kinetic

data are presented have accompanying KIE data in Table 3.

Table 3. Kinetic Isotope Eﬀects for PAS-Catalyzed

Hydrolysis of Sulfate Monoester 1d and Phosphate

Monoester 2d

The leaving group KIEs ¹⁵(V/K) and ¹⁸(V/K)_bridgereport on

how leaving group stabilization might be compromised by

mutation. Loss of general or Lewis acid catalysis will result in

increases in both of these KIEs relative to the native enzyme.

The isotope eﬀect on the nonbridging oxygen atoms, ¹⁸(V/

K)_nonbridge, monitors the hybridization state of the transferring

sulfuryl or phosphoryl group, which aﬀects the P−O or S−O

nonbridging bond orders and hence their vibrational frequen-

cies. A loose transition state gives rise to slightly inverse eﬀects as

these bond orders increase. This isotope eﬀect becomes

increasingly normal (i.e., approaching or exceeding a value of

1) as the transition state grows more associative in nature.

The small ¹⁵(V/K) of 1.0006 (Table 3), for PAS WT-

catalyzed hydrolysis of sulfate monoester 1d suggests nearly

complete neutralization of the negative charge developing on the

leaving group from S−O bond ﬁssion. Similar neutralization

occurs by intramolecular pre-equilibrium proton transfer during

the uncatalyzed hydrolysis of neutral sulfate monoesters under

acidic conditions⁷⁸(¹⁵(V/K) = 1.0004). The ¹⁸(V/K)_bridgeKIE

for the PAS-catalyzed reaction is similar to that of the

uncatalyzed hydrolysis of the neutral monoester⁷⁸and the AP-

catalyzed sulfate monoester hydrolysis²¹(Table 3, Figure 4a). In

both cases signiﬁcant masking of leaving group charge

development occurs due to intramolecular proton transfer or

interaction with a Lewis acid, respectively. The magnitude of

¹⁸(V/K)_bridgeis consistent with signiﬁcant S−O_bridgebond ﬁssion

concomitant with leaving group stabilization, either by

interaction with a positively charged group (Lewis acid) or

protonation. A similar observation has been made for phosphate

¹⁸(V/

substrate

catalyst

solution

¹⁵(V/K)

¹⁸(V/K)_bridge

K)_nonbridge

1d

1.0004 (1)

1.0101 (2)

1.0098 (3)

a

(neutral)

solution

(monoanion)

WT

1.0026 (1)

1.0210 (10)

0.9951 (3)

b

1.0006 (4)

1.0012 (6)

1.0013 (2)

1.0088 (9)

1.0097 (19)

1.0216 (17)

1.0064 (15)

1.0039 (4)

1.0004 (6)

C51S

H115A

H211A

K375A

c

1.0023 (5)

1.0004 (2)

1.0244 (23)

1.0087 (3)

0.9958 (11)

1.0184 (5)

2d

solution

(monoanion)

d

solution

(dianion)

1.0028 (2)

1.0189 (5)

1.0102 (9)

0.9994 (5)

0.9912 (8)

d

WT

1.0007 (4)

Recorded in 10 N HCl, 15 °C.⁷⁸Recorded at pH 9.0, 85 °C.⁷⁸

a

b

c

d

Activity too low to be determined. Recorded at 95 °C.⁵³

Because the KIEs were measured by the internal competition

method, they describe eﬀects on k_cat/K_M(or V/K),⁹⁴

encompassing steps from free enzyme and substrate up to the

ﬁrst irreversible step, regardless of which step in the full

mechanism is rate-limiting. In the present case, as described

above, this is representative of the rate constant for the ﬁrst

chemical step (k₂in Figure 1c). Thus, the reported KIEs reﬂect

the TS for the substrate reacting with the formyl glycine

nucleophile, even though the overall rate-determining step is

breakdown of the intermediate.

H

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Figure 4. KIEs for PAS-catalyzed hydrolysis of sulfate monoester 1d and phosphate monoester 2d. The values are listed in Table 3 (PAS and solution

data) and Table S15 (all other enzymatic data, Supporting Information). (a) The ¹⁸O KIEs for the bridging (or leaving group) oxygen. (b) The ¹⁸

O

KIEs for the nonbridging oxygens. The dotted lines indicate extremes of ¹⁸O KIEs for tight (¹⁸O bridge = 0%, ¹⁸O nonbridge = +2.5%) and loose (¹⁸O

bridge = +3.0%, ¹⁸O nonbridge = −0.5%) transition states.⁸⁰AP: E. coli alkaline phosphatase,²¹PP1: Protein Phosphatase 1,⁵⁸PTPase: Yersinia Protein

Tyrosine Phosphatase.⁵⁴

monoester hydrolysis catalyzed by AP,²¹protein phosphatase

1⁵⁸(PP-1), and several protein-tyrosine phosphatases

(PTPs)^{51,52,54,55,97}(Figure 4a, Table S15).

hydrolysis of the 1d monoanion⁷⁸(−0.49%). This value is closer

to the ¹⁸(V/K)_nonbridgefor the hydrolysis of the neutral sulfate

monoester⁷⁸(+0.98%). However, in this case, the normal KIE

arises from deprotonation of the sulfuryl group (i.e., proton

transfer from S−O−H to the leaving group). In previous

investigations of the TS for enzymatic sulfate and phosphate

transfer the drop in the normal ¹⁸(V/K)_bridgewas accompanied

by a more inverse²¹or mostly unchanged ¹⁸(V/K)_nonbridge

KIE^{51,52,54−56,58,97}(compared to the ¹⁸(V/K)_nonbridgefor

uncatalyzed phosphate dianion hydrolysis; i.e., ≤0.9994), and

suggests that the TS in these enzymatic reactions is still largely

In the PAS WT-catalyzed hydrolysis of phosphate monoester

2d, the ¹⁵(V/K) and ¹⁸(V/K)_bridgeKIEs also indicate near

complete neutralization of the charge on the leaving group and

signiﬁcant P−O_bridgebond ﬁssion (Table 3, Figure 4a). The

¹⁸(V/K)_nonbridgeKIE for PAS WT-catalyzed hydrolysis of

phosphate monoester 2d was more inverse than for the

uncatalyzed solution reaction of the dianion (0.9912 vs

0.9994; Table 3, Figure 4b) and is similar to the value for its

hydrolysis by the superfamily member AP R166S (0.9925).³⁶In

the case of AP the inverse shift in this KIE was attributed to

interactions of the phosphoryl group with the metal ions and

hydrogen-bonding residues at the active site. In the PAS reaction

similar interactions are possible (Figure 1). For the PAS WT-

catalyzed hydrolysis of sulfate monoester 1d the ¹⁸(V/K)_nonbridge

is normal (1.0064, or +0.64%, Table 3 and Figure 4b), in

contrast to the inverse ¹⁸(V/K)_nonbridgefor the uncatalyzed

obs

dissociative despite the less negative β_{leaving group}and drop in

normal ¹⁸(V/K)_bridgeKIE compared to the uncatalyzed reaction,

which arise from neutralization of the charge developing on the

leaving group.

The normal ¹⁸(V/K)_nonbridgeis unlikely to arise from the same

origin as in the uncatalyzed hydrolysis of the neutral sulfate

monoester, since protonation of the sulfuryl group oxygens will

not occur, except under extremely acidic conditions. A more

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plausible explanation is that the PAS WT-catalyzed TS is more

associative than the solution reaction of the monoanion. The

latter scenario could also partly explain the unexpectedly large

the leaving group oxygen in the TS. Direct proton transfer

involving lysine as the donor in enzyme active sites is rare, and

charge compensation by Lewis acid interaction with the cationic

charge of the proton shared between H211 and K375 is a

potential alternative. The PAS WT-catalyzed sulfate monoester

hydrolysis in D₂O is more sensitive to the leaving group

(β_{leaving group}is more negative) compared to the same reaction in

H₂O (Figure S12). The observed diﬀerence in leaving group

eﬀect of the removal of residues that only interact indirectly with

obs

the nonbridging leaving group oxygen on β_{leaving group}

.

The PAS K375A mutation results in a reaction for which the

¹⁵(V/K) and ¹⁸(V/K)_bridgeKIEs are largely identical to those of

the uncatalyzed hydrolysis (Table 3, Figure 4b), suggesting this

residue is largely responsible for leaving group neutralization in

the TS. This is consistent with the eﬀect of the mutation on the

β_{leaving group}^obsand similar to the value observed for the removal of

an aspartic acid performing a similar role in PTPs.^52,54,55The

data suggest that this residue either protonates the leaving group

directly or that its mutation results in a dislocation of H211

(Figure 1), interfering with its function in this role, implying

synergy between these two residues, as discussed earlier. The

¹⁸(V/K)_nonbridgefor PAS K375A is identical to the uncatalyzed

hydrolysis, which could be explained by the loss of coordination

of K375 to the sulfuryl group.

dependence (Δβ_{leaving group}^H‑D) is +0.06

0.03, which

corresponds to a k_H/k_Dratio ranging from ∼1.7 at pK_a^{leaving group}

= 5.5 to ∼3.3 at pK_a^{leaving group}= 10. These data suggest that, with

increasing demand for leaving group stabilization, a proton

transfer event becomes more rate-limiting. This would suggest

that the degree of S−O_bridgebond ﬁssion during the TS is

reduced with increasing leaving group ability (i.e., S−O_bridge

bond ﬁssion is almost complete prior to proton transfer for the

low pK_aleaving groups that require charge oﬀset assistance less).

However, the ¹⁵N KIE for PAS WT-catalyzed hydrolysis of

sulfate monoester 1d (pK_a= 7.02) suggests almost complete

charge compensation on the leaving group oxygen, suggesting

K375 is mainly responsible for charge compensation without

transferring its proton during catalysis. For PAS K375A, there is

The relatively modest eﬀect of the C51S mutation on ¹⁵(V/K)

and ¹⁸(V/K)_bridgecompared to the wild-type reaction is

consistent with the modest eﬀect of this mutation on

β_{leaving group}^obs. As stated above, the TS of the PAS WT-catalyzed

sulfate monoester hydrolysis has a more associative character

than in the uncatalyzed reaction; that is, the nature of the

nucleophile is thought to be more important, based on the

change to a normal ¹⁸(V/K)_nonbridge. However, the change in

nucleophile from fGly to serine has a much smaller eﬀect on

¹⁸(V/K)_nonbridgethan the removal of K375, which is thought to

interact directly with the nonbridging oxygens (Figure 1).

The mutation of H115 to alanine has a large eﬀect on the KIEs

compared to WT, despite the absence of direct TS interaction

between H115 and the substrate (Table 3, Figure 4). The ¹⁸(V/

K)_bridgeis nearly as large as that observed for PAS K375A. The

¹⁵(V/K) shows partial charge neutralization on the leaving

group, intermediate between the WT reaction and that of

K375A. A possible explanation is suboptimal orientation of the

substrate relative to the residues mainly responsible for leaving

group stabilization, K375 and H211. The reaction of the H115A

mutant shows a ¹⁸(V/K)_nonbridgethat is also intermediate

between the WT and K375A, also consistent with less than

optimal coordination of the nonbridging oxygens to K375. The

fact that mutation H115A has a larger eﬀect on ¹⁸(V/K)_nonbridge

than C51S conﬁrms that the large change in ¹⁸(V/K)_nonbridgefor

the PAS WT-catalyzed reaction compared to the sulfate

monoester monoanion uncatalyzed hydrolysis is most likely

not dependent on the nucleophile but the result of interactions

between the nonbridging oxygens and positively charged

functional groups (K113, K375 and Ca²⁺). The nature of the

contribution of the latter interactions to TS stabilization appears

to be unique for PAS WT-catalyzed sulfate monoester

hydrolysis, since the eﬀect of the enzyme on the ¹⁸(V/K)_nonbridge

for phosphate monoester hydrolysis is completely diﬀerent.

However, both reactions show a similar degree of leaving group

stabilization.

obs

no diﬀerence in β_{leaving group}when recorded in H₂O or D₂O

(Figure S13). However, a pK_a-independent k_H/k_Dof ∼2 for all

reactions was observed, suggesting that a leaving group

independent proton transfer event is rate-limiting for this

mutant. The solvent isotope eﬀect is consistent with the

suggested catalytic role for K375 as the main residue responsible

for leaving group stabilization during bond breaking in the TS,

since any leaving group-dependent proton transfer event will

most likely be fast compared to the severely slowed S−O_bridge

bond ﬁssion. Indeed, the heavy-atom isotope eﬀects suggest that

these two events occur in the same step.

IMPLICATIONS AND CONCLUSIONS

■

Assignment of Catalytic Roles to Active-Site Residues.

A mechanistic pathway for PAS WT-catalyzed sulfate monoester

hydrolysis (Figure 1b) had been suggested by structural analysis

but can now be ﬁrmly established on the basis of the results

presented in this study, including a reassessment of the

contributions of the active-site residues. The presence of nine

highly interconnected polar residues and a divalent metal cation,

all of which could contribute to catalysis, makes assignment to

speciﬁc function necessarily diﬃcult, even when a high-

resolution structure is available and patterns of amino acid

conservation can be discerned.⁹⁸The importance of each of

these residues was further established in mutational scanning

experiments with hASA (by changing them to either asparagine

or aspartate),⁴⁹but the observed v_maxdecreases and K_Mincreases

implied that each of them was critical, leaving an ambiguity

about their respective roles. An abundance of potentially

functional residues is frequently observed. Indeed, such

abundance has been convincingly explained by Kraut et al.⁹⁹

to be a result of the cooperativity of active sites that prevents a

simple quantitative assignment of mechanistic contributions to

individual residues. This present work exempliﬁes how LFERs

can be employed to probe the role of catalytic residues

comparatively in mutants, providing insights that amino acid

scanning experiments evaluated by measurement of Michaelis−

Menten parameters for one substrate do not provide. For

example, leaving group stabilization by the proposed general

acid H211 is much less important than that of K375 (based on

the much smaller eﬀect on β_{leaving group}^obsupon alanine scanning).

Leaving Group Dependence in D₂O. The considerably

less negative β_{leaving group}^obsand a ¹⁵N KIE near unity for the PAS

WT-catalyzed sulfate monoester hydrolysis could be caused by

protonation of the leaving group oxygen, as in PTPs.^{52,54−56,59}

Lewis acid charge neutralization by metal ions can have the same

eﬀect.²¹The available data for PAS all point to K375 as the main

residue responsible for stabilization of charge that develops on

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Figure 5. Comparison of mechanisms of leaving group stabilization in the transition states of sulfate monoester hydrolysis by PAS and phosphate

monoester hydrolysis by alkaline phosphatase. (a) During ﬁssion of the sulfate ester bond negative charge develops on the leaving group and is oﬀset by

a proton, held by the H211−K375 pair. (b) In EcAP^26,29the charge developing on the leaving group as a serine nucleophile attacks is oﬀset by a metal

ion. (c) Structural superposition of PAS⁴⁴(pdb entry 1HDH) and AP¹⁰⁰(3TG0), including bound product in the active site (inorganic sulfate and

phosphate, respectively. The functionally homologous residues align well, showing that the second divalent metal ion (Zn²⁺) of AP is providing charge

oﬀset in a similar position as the proton held by H211 and K375 in PAS.

The LFERs serve to measure the eﬀective charge “seen” at the

leaving group oxygen on moving from ground to transition

state¹⁰⁵and quantify the charge compensation achieved by the

enzyme via its side chains: in the absence of a charge-

compensating residue a larger charge is seen at this oxygen.

The combined eﬀect of removing both these residues was larger

than the sum of its eﬀects in wild-type enzyme, suggesting a high

degree of interdependence between them with regard to leaving

group stabilization, possibly by sharing a proton (Figure 5). The

LFERs for k_catand 1/K_Mfor PAS WT-catalyzed sulfate

monoester hydrolysis and the pre-steady-state measurements

with sulfate monoester 1d suggest that the leaving group-

dependent sulfate ester bond ﬁssion (S−O_bridgeﬁssion; step 1 in

Figure 1b) is much faster than the cleavage of the hemiacetal

intermediate (step 2, Figure 1b).

the substrate for optimal interaction with K375. (iv) Structural

alignment of PAS⁴⁴with AP¹⁰⁰shows that K375 occupies a

similar position as the divalent metal ion thought to provide

leaving group stabilization in AP (Figure 5).

Compensation of charge development on the leaving group

oxygen by an amino acid side chain may be expected to involve

direct protonation of the leaving group oxygen. The cleavage of

this S−O_bridgeester is dependent on, and occurs in concert with,

a near-complete neutralization of the charge on the leaving

group for sulfate monoester 1d (pK_a^{leaving group}= 7.03), as

evidenced by a ¹⁵N KIE near unity (Table 3). This would

suggest near-complete proton transfer during the TS, but

transfer of the proton would only occur once the S−O_bridgebond

cleavage is well advanced, if at all (Figure 5a,b). Charge

compensation at the leaving group oxygen is nearly complete for

PAS WT-catalyzed hydrolysis of sulfate monoester 1d. As the

proton is shared between H211 and K375 (Figure 5a,b) it may

be that actual proton donation from lysine does not take place,

and instead transfer to H211 results in the observed

compensation. Comparison of the leaving group dependences

in H₂O and D₂O showed an increasing k_H/k_Dratio for k_cat/K_M

with decreasing leaving group ability (Figure S12, Supporting

Information), suggesting that proton transfer is becoming

increasingly more important for S−O_bridgeester bond ﬁssion.

The absence of the leaving group-dependent change in k_H/k_Dfor

PAS K375A (Figure S13) further supports the importance of

K375 for S−O_bridgebond ﬁssion, since it is in agreement with S−

O_bridgeester bond ﬁssion being fully rate-limiting for leaving

group departure in this mutant. (This solvent isotope eﬀect is

relevant for the ﬁrst chemical step, while the isotope eﬀect on k_cat

reﬂects the overall rate-determining step, i.e., breakdown of the

intermediate).

The strongest eﬀect on leaving group-dependent catalysis was

seen for the K375A mutants (Δβ_{leaving group}^obs= +0.61, Figure 2f,

Table 2), further underlining that K375 is the most important

residue for stabilization of the negative charge that develops on

the leaving group oxygen during catalysis. This conclusion was

also supported by the following observations: (i) The ¹⁵N and

18

O

KIEs for mutant K375A are essentially the same as for

bridge

the solution reaction. The wild type showed almost complete

charge compensation on the leaving group (¹⁵N KIE essentially

unity) and a lowered normal ¹⁸O_brigdeKIE (Table 3, Figure 4).

(ii) For PAS K375A the leaving group-dependent step becomes

increasingly more rate-limiting with decreasing leaving group

ability (Figures 2f and S10b), pointing to its involvement in

leaving group stabilization. (iii) The unexpectedly large eﬀects

obs

on β_{leaving group}for active site mutants C51S and H115A are

dependent on the presence of K375 (Table 2), suggesting that

the main function of the other active-site residues is to position

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Figure 6. PAS variants with lower catalytic eﬃciencies are more sensitive to the leaving group ability. (a) Correlation between catalytic eﬃciency

(represented by log[k_cat/K_M]) and leaving group dependency (β_{leaving group}^obs) of the various PAS variants (closed symbols: PAS WT and its single

mutants, open symbols: double mutants) for 3-nitrophenyl sulfate 1e. Linear correlations including all variants (red line, R²= 0.79; p = 0.0033) and

only based on the single mutants and wild type (i.e., excluding the open symbols; blue line, R²= 0.83; p = 0.011) show slopes of 10.8 2.3 and 9.1

2.0, respectively. (b) Correlation between the response slope as shown in (a) and pK_avalues of the substrate’s leaving group (see Figure S14 for the

corresponding red and blue correlation lines from which the slopes were obtained). The slopes for sulfate monoesters 1f and 1k (open blue symbols)

clearly deviate from the observed trend (suggesting idiosyncratic active-site interactions) and were therefore not included in the ﬁt (see Figure S15,

Supporting Information, for a ﬁt that includes all data points). Red ﬁt: slope: 1.16 0.05; R²: 0.99; p: 2.2 × 10⁻⁴. Blue ﬁt: slope: 1.0 0.06; R²: 0.97; p:

<1 × 10⁻⁴.

An alternative mechanistic scenario in which additional steps

prior to the ﬁrst irreversible S−O_bridgeester bond ﬁssion are rate-

limiting for wild type, but the chemical step becomes limiting in

a mutant, may be considered. Such an explanation has been

advanced for FLAP endonuclease, where pH-rate proﬁles

suggested rate-limiting physical steps after substrate binding

resulted in a commitment to catalysis that suppressed the

magnitude of β_{leaving group}^obsand was reduced or eliminated by a

mutation that slowed the chemistry step.¹⁰¹However, this

scenario is inconsistent with the observed KIEs on PAS catalysis.

A commitment factor would reduce all the observed KIEs in

equal proportion.^94,102Thus, if making the chemical step more

rate-limiting (reducing a commitment factor) was the cause of

the larger Brønsted slope, the KIEs would increase in the same

proportion. Instead, the K375A mutant reaction gives a ¹⁵N KIE

that is fourfold higher than wild-type PAS; the bridge ¹⁸O eﬀect

is 2.8-fold greater; and the nonbridge ¹⁸O eﬀect goes from

normal to inverse. The substrates in FLAP and PAS also show

signiﬁcant diﬀerences: DNA substrates of FLAP may require

unpairing or helical arch ordering, whereas for the small-

molecule substrates of PAS no analogous steps are conceivable.

In addition, the high K_Mvalues are consistent with reversibility

and a rapid equilibrium between bound and unbound states

(contrasting with the nanomolar binding in FLAP). The LFERs

and KIEs for PAS use k_cat/K_Mcomparisons that reﬂect the ﬁrst

irreversible step of the reaction, which thus is unlikely to be

associated with binding.

phosphatases the small inverse ¹⁸O_nonbridgeKIE was either more

inverse²¹or virtually unchanged^{51,52,54,55,58,97}(Figure 4b, Table

S15). The possible change from a dissociative to a more

associative TS does not apply to all PAS-catalyzed conversions,

since the promiscuous enzyme-catalyzed hydrolysis of phos-

18

phate monoester 2d showed a more inverse

O

.

nonbridge

Enzymatic speciﬁcity toward sulfate over phosphate monoester

cannot be primarily based on overall charge, demand for leaving

group stabilization, or nucleophile strength, since these either

provide no discrimination or will favor the more highly charged

phosphate monoesters. The diﬀerence in eﬀect on the

18

O

nonbridge

KIE suggests that the subtle diﬀerences in geometry

between the S−O_nonbridgeand P−O_nonbridgebonds are responsible

for making PAS speciﬁc toward sulfate monoesters.

Relationship to Computational Studies. The existence

of computational studies attempting to model aspects of PAS

catalysis in silico^38,42,43provides an opportunity for a comparison

of their conclusions with our experimental results. One report

has taken a shortening of the S−O_lgbond distances (i.e., the

18

O

bridge

in our study) as an indication for a shift to a more

associative transition state.³⁸However, these distances are not

equivalent to shifts in TS. We ﬁnd that the ¹⁸O_bridgeKIE is similar

in both sulfate monoesters and phosphate monoesters, yet the

nonbridging oxygen data (for which no computational results

are available) diﬀer widely. Importantly, only limited informa-

tion on the individual contributions of active-site residues has

been provided by computational studies. The residue that we

show is central to both leaving group stabilization and shifting

the nature of the TS for PAS-catalyzed sulfate monoester

hydrolysis, K375, has not been discussed in the context of

quantum mechanics/molecular mechanics (QM/MM) studies.

Instead, Marino et al.⁴²highlight the eﬀect of H211 but assign its

role as the sole general acid catalyst, while no such eﬀect is

ascribed to K375. Here, and in the work of Luo et al.,³⁸residues

thatin our analysisare assigned only indirect roles aﬀecting

the nature of the TS and leaving group stabilization (K113,

Although the exact residues and functional groups involved

diﬀer between several previously characterized phospha-

tases^{21,23,24,33,36,51,52,54−56,58,59,97}and PAS, all these enzymes

rely on leaving group stabilization by charge compensation,

obs

resulting in less negative β_{leaving group}values and lowered, but

18

still normal,

O

KIEs. However, the KIEs for PAS are

bridge

consistent with a more associative mechanism than previously

reported phosphatases, because of the observed change of

18

O

KIE from inverse (−0.49%) to normal (+0.62%)

nonbrigde

compared to the solution reaction (Table 3, Figure 4b). In

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H115, fGly51) feature more prominently. A third study⁴³uses

molecular dynamics simulations (based on active-site group

ground-state pK_avalues validated by PROPKA) to deﬁne a

model that features K375 but not His211.

enzyme catalysis¹⁰⁴by providing a quantitative picture of elusive

transition-state interactions, through the measurement of

eﬀective charge changes via Brønsted plots.¹⁰⁵These, combined

with isotope eﬀects studies, are applied here systematically for

the ﬁrst time to studies of mutational eﬀects to quantitatively

assess the individual involvement of particular residues in PAS

catalysis.

In computational studies with AP diﬀerent authors obtained

diverging predictions for enzymatic TSs that were either more

associative⁴⁰or more dissociative.²⁷It is possible that the

calculated relatively ﬂat calculated energy surfaces (e.g., as

shown in refs 39 and 103) make precise assignment of a TS

position in More O’Ferrall/Jencks-type diagrams diﬃcult in

silico, as many energetically near-equivalent paths of bond-

making and breaking seem to exist, yet these ambiguities may

also be artifacts of the computational methods. Experimentally

the notion of ﬂat calculated energy surfaces is not supported:

even in promiscuous enzymes the nature of the TS is close to

that in solution,^23,32,36,37suggesting it is hard to change.

Computational results from modeling physical processes are not

direct experimental observations and should not be used as

substitutes of experimental reality. A more conﬁdent assignment

of the nature of the TS requires resolution by experimental

evidence of the sort presented here, that is, a kinetic analysis

involving juxtapositions of LFERs and KIEs.

In comparing experimental and computational results it is

important to bear in mind that in in silico work ground-state

binding eﬀects (contributing to K_M) are excluded and that

elementary rate constants are calculated that may be implicit in

Michaelis−Menten parameters but hard to disentangle. Multiple

potentially rate-limiting steps may make these parameters

complex functions of rate constants, preventing straightforward

reconciliation of data. In some cases the computed energy levels

do not reproduce the actually measured rates, indicating

possible shortcomings in capturing the solution reaction,¹⁰³

but in the case of enzymatic reactionsalso leading to

postulation of diﬀerent rate limiting steps. For example, the

rate-limiting step of the PAS reaction had been predicted to be

k₂,⁴²which our analysis suggests to be incorrect. Our stopped-

ﬂow data suggest that k₂(i.e., the relevant step in the LFERs) is

at least 100-fold faster than k_cat. All these factors necessitate the

judicious evaluation of both experimental and computational

data with respect to functional conclusions, with many aspects

necessitating experimental conﬁrmation or resolution.

A Quantitative Measure for the Eﬃciency of General

Acid or Charge Compensation Catalysis. The correlation of

reaction rates and the measured values for β_{leaving group}^obs(Figure

6) establishes a direct link between catalytic eﬀects by charge

compensation (and/or proton transfer) and catalytic eﬃciency:

the more the catalytic eﬀect of the protonation of an active site

group is removed (as indicated by larger eﬀective charge changes

at the leaving group oxygen), the more the overall rate suﬀers.

This eﬀect is more pronounced for unreactive substrates that

require interaction with the proton held by K375 and H211 to a

greater extent (Figure S15), such as the presumed natural

substrates, sulfated sugars.

The correlation of catalyst and substrate reactivity provides a

further measure of sensitivity of catalysis to substrate reactivity

changes and should be used to quantitatively assess the

eﬃciency of general acid catalysis. In the future it will be

interesting to contrast slopes in such double reactivity plots (as

in Figure 6b) for several enzyme systems and use its slopes to

quantify the sensitivity of catalytic eﬀects in diﬀerent active site

arrangements.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.bio-

chem.8b00996.

Synthetic procedures, mathematical descriptions for

enzyme kinetics and expected detection limits, determi-

nation of kinetic isotope eﬀects, and data analysis.

Michaelis−Menten plots for sulfate substrates hydrolyzed

by PAS WT, tables of experimentally measured steady-

state kinetic parameters, additional LFERs, stopped ﬂow

data, and primers used for mutant construction (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: fh111@cam.ac.uk. (F.H.)

*E-mail: alvan.hengge@usu.edu. (A.C.H.)

■

ORCID

Bert van Loo: 0000-0003-4253-2163

Alvan C. Hengge: 0000-0002-5696-2087

Florian Hollfelder: 0000-0002-1367-6312

Present Addresses

^§Institute for Evolution and Biodiversity, University of Mu

̈

nster,

Germany.

^∥Faculty of Tropical Medicine, Mahidol University, Thailand.

^⊥Institute of Biochemistry, Faculty of Medicine, University of

Ljubljana, Slovenia.

Funding

This research was funded by the Biological and Biotechnological

Research Council (BBSRC, BB/I004327/1) and the Engineer-

ing and Physical Sciences Research Council (EPSRC, EP/

E019390/1) and NIH grant GM 47292 to A.C.H.; F.H. is an

ERC Investigator (695669); U.B. received a studentship from

the Royal Thai Government; and M.G. was supported by a

postdoctoral Marie-Curie fellowship from the EU.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We thank M. Hyvonen for his help in the preparation of

structural ﬁgures.

■

ABBREVIATIONS

■

AP, alkaline phosphatase; LFER, linear free energy relationship;

KIE, kinetic isotope eﬀect; PAS, Pseudomonas aeruginosa

arylsulfatase.

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