The role for glutamic acid at position 196 in human hypoxanthine phosphoribosyltransferase (HPRT) as investigated using site-directed mutagenesis

Article abstract of DOI:10.1080/15257770802146593

The crystal structure of human HPRT reveals the involvement of E196 side chain at the A-B dimer interface. Interference by valine substitution at this position (E196V), as identified in patients with Lesch-Nyhan disease, nearly abolishes enzymatic activity. Kinetic analysis of the active mutants (E196A, E196D, E196Q, and E196R) suggests that interaction between K68 and E196 side chains contributes to stabilization of cis-configuration during the catalytic cycle. The study also provides further insight into the role of A-B dimer interactions relating to K68 in the regulation of cis-trans isomerization that potentially governs the rate-limiting steps in the HPRT reaction. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802146593

Nucleosides, Nucleotides, and Nucleic Acids, 27:894–899, 2008
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802146593
THE ROLE FOR GLUTAMIC ACID AT POSITION 196 IN HUMAN
HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE (HPRT) AS
INVESTIGATED USING SITE-DIRECTED MUTAGENESIS
B. Canyuk,1 A. E-Wan,1 W. Keawwijit,1 T. Nualnoi,1 L. Sirisatean,1
P. Tansakul,2 and C. Tanthana1
1Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Science, Prince of
Songkla University, Songkhla, Thailand
2Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical
Sciences, Prince of Songkla University, Songkhla, Thailand
The crystal structure of human HPRT reveals the involvement of E196 side chain at the A-B
2
dimer interface. Interference by valine substitution at this position (E196V), as identified in patients
with Lesch-Nyhan disease, nearly abolishes enzymatic activity. Kinetic analysis of the active mutants
(E196A, E196D, E196Q, and E196R) suggests that interaction between K68 and E196 side chains
contributes to stabilization of cis- configuration during the catalytic cycle. The study also provides
further insight into the role of A-B dimer interactions relating to K68 in the regulation of cis- trans
isomerization that potentially governs the rate-limiting steps in the HPRT reaction.
Keywords Human HPRT; LND; glutamic acid 196; E196, functional role
INTRODUCTION
Hypoxanthine phosphoribosyltransferase (HPRT, E.C. 2.4.2.8) is a key
enzyme in the purine salvage pathway. HPRT catalyzes the reversible trans-
fer of a phosphoribosyl moiety from phosphoribosylpyrophosphate (PRPP)
to 6-oxopurine bases. The enzyme exhibits a sequential kinetic mechanism
with PRPP binding first followed by the purine base. After catalysis, py-
rophosphate (PPi) is released before the nucleotide. The releases of nu-
cleotide and PRPP are rate-limiting steps in the nucleotide formation and
pyrophosphorolysis, respectively.[1] Human HPRT in the crystal structures
exists as either a dimer or a tetramer, consistent with a previous report.[2]
The A-B dimer interface is conserved in both dimer and tetramer.[3] The
This research project was supported by a grant from Prince of Songkla University.
Address correspondence to B. Canyuk, Department of Pharmaceutical Chemistry, Faculty of
Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand. E-mail:
bhutorn.c@psu.ac.th
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Role for E196 in Human HPRT
895
FIGURE 1 Noncovalent interactions (dashed lines) between residues in the active sites of human HPRT
with bound substrates and residues in the opposing subunit (V96 and D97) at the A-B dimer interface,
as deduced from the crystal structure.[4]
K68 of the active site loop I participates in dimer formation. Loop I is
highly conserved as a tripeptide of L67-K68-G69. The peptide between L67-
K68 adopts non-proline cis-configuration in the crystal structures of hu-
man HPRT with fully occupied active site.[4] The cis-geometry enables the
K68 side chain to extend across the A-B dimer interface and interacts with
E196 of the same subunit and V96 and D97 of the opposing subunit. Si-
multaneously, cis-geometry allows the K68 main-chain hydrogen to interact
with the PPi moiety (Figure 1). However, the L67-K68 peptide adopts trans-
configuration in the apo-enzyme which allows the K68 side chain to interact
with the re-oriented E133 and D137 that blocks the binding sites of PRPP
and 5’-phosphoribosyl portion of nucleotides.[5]
The cis-trans isomerization of HPRT has been proposed to regulate
substrate binding and product release throughout the catalytic cycle.[6]
The involvement of K68 at the A-B dimer interface is proposed to pro-
vide communication between subunits. The molecular mechanism for such
communication in the catalysis is still unclear. Interference of the native mi-
croenvironment of the K68 side chain, such as valine substitution at the po-
sition 196 (E196V), leads to Lesch-Nyhan disease (LND) in humans.[7] This
study is aimed to clarify the functional contribution of E196 in HPRT catal-
ysis by site-specific mutagenesis and functional analyses using a functional
complementation assay and steady state kinetic analysis. Understanding the
contribution of E196 would provide further insight into the functional role
of A-B dimer interactions relating to K68.

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B. Canyuk et al.
MATERIALS AND METHODS
Construction of Recombinant Plasmids Harboring cDNA
Encoding Human WT-HPRT and E196 Mutant HPRTs
The cDNA of WT-HPRT was prepared by RT-PCR using specific primers
and mRNA extracted from human white blood cells. The cDNA was inserted
into pBAce plasmid.[8] The mutagenic cDNAs encoding E196A, E196D,
E196Q, E196R, and E196V HPRTs were prepared by overlap extension using
PCR with mutagenic primers and inserted into pBAce plasmid.[9]
Functional Complementation Assay
Catalytic activity of each mutant was individually assessed by measuring
growth of purine auxotroph Escherichia coli SØ609 transformants expressing
the recombinant mutant HPRT, in semi-defined liquid media supplemented
with hypoxanthine and guanine at 37◦C.[10] The growth curves were plotted
between incubation time (hours) versus OD600 of cultures.
Expression and Purification of Recombinant HPRTs
Recombinant WT and E196 mutant HPRTs were expressed in E. coli
SØ606 transformants in low phosphate induction media.[10] The WT HPRT
was purified by affinity chromatography with GMP-agarose (Sigma, St. Louis,
MO, USA). Mutant HPRTs could not be purified by affinity chromatography
because they bound very weakly to GMP-agarose. They were isolated from
crude lysates by heating at 80◦C. After heating, the soluble fractions contain-
ing catalytically active mutant HPRT were further purified by size exclusion
chromatography.
Determination of Steady State Kinetic Parameters of WT
and Active E196 Mutant HPRTs
Kinetic parameters for the nucleotide formation and pyrophosphoroly-
sis were measured by spectrophotometric methods.[1] The Km and kcat values
were determined by non-linear regression analysis using the KaleidaGraph
software, version 3.5b5 (Synergy Software, Reading, PA, USA).
RESULTS
Functional Complementation Assay
Growth rates of E. coli SØ609 transformants reflect the activity of recom-
binant HPRT.[10] Growth rates of those cultures expressing the recombinant
WT and all mutant HPRTs except E196V were similarly exponential and

Role for E196 in Human HPRT
897
reached stationary phase with OD600 values of 0.6–0.7, within 10–12 hours.
In contrast, the growth rate of the E. coli SØ609 transformant expressing
the recombinant E196V mutant HPRT was extremely slow and OD600 value
reached only 0.05 after incubation for 10 hours (results not shown).
Binding Affinity of Recombinant WT and E196 Mutant HPRTs
to GMP-Agarose
The WT HPRT was purified to near homogeneity by using its high affin-
ity to GMP-agarose. Affinities to GMP-agarose of all E196 mutants were very
low (results not shown). HPRT is highly thermostable, so all highly active
E196 mutants could be partially purified from bacterial crude lysates by
heating at 80o C. The mutant HPRTs constituted major component in the
soluble fractions and their activities were retained. Subsequent purification
of the soluble fractions by size-exclusion chromatography provided highly
purified mutant HPRTs.
Steady State Kinetic Parameters
Steady state kinetic parameters for nucleotide formation and pyrophos-
phorolysis of WT HPRT and highly active E196 HPRT mutants are sum-
marized in Table 1. Kinetic parameters of the E196V mutant were not
determined due to extremely low enzyme activity.
DISCUSSION
Removal of the carboxylate side chain at position 196 by alanine substitu-
tion (E196A) or replacement with polar or charged residues at position 196
(E196D, E196Q, and E196R) did not affect the activity of mutant HPRTs, as
demonstrated in a functional complementation assay. This result indicated
that E196 did not directly participate in the reaction chemistry. However,
substitution at E196 with bulky and hydrophobic residue (E196V) resulted
in marked decrease of the activity and is consistent with the finding that
the E196V mutation in human leads to LND. The E196 side chain together
with V96 and D97 in the opposing subunit (Figure 1), potentially stabilize
K68 in the cis-configuration. Thus, removal of the cis-stabilizing contribu-
tion by E196 mutations likely enhances propensity for trans-isomerization.
The trans-configuration apparently obstructs the binding site of PRPP and
5ꢁ-phosphoribosyl portion of IMP which is consistent with marked increase
in Km values of all substrates (6-432 fold of WT) except hypoxanthine, for
mutant HPRTs. The large increase in kcat for mutants (4 to 13-fold of WT)
is likely the result of the marked increase in Km values of products (IMP
or PRPP), whose releases exert rate-limiting steps in the WT enzyme. In-
crease in the Km of IMP is consistent with low affinity of all mutants to

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Role for E196 in Human HPRT
899
GMP-agarose. Weakened stabilization of cis-configuration in the K68R HPRT
of Trypanosoma cruzi also resulted in marked decrease in affinity for IMP
(280-fold Km of WT and >20-fold KD of WT) and GMP.[11] Weakened trans-
stabilization is potential in the D137A HPRT of T. cruzi which imposed no
effect on Km for hypoxanthine but resulted in 22-fold reduction in the Km
for PRPP.[10]
The result presented herein suggests the role of A-B dimer interactions
relating to K68 in the regulation of cis-trans isomerization that governs the
rate-limiting step in the HPRT reaction. Valine substitution (E196V) almost
abolished the enzymatic activity, indicating that the mutation imposed a
more deleterious structural consequences that needs to be further clarified.
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