16120 J. Phys. Chem. B, Vol. 114, No. 49, 2010
Ruzicka and Frey
TABLE 1: 2,3-Aminomutase Activities of Lysine
min, 10-35% B in 17.5 min, and 35-100% B in 15 min. PITC-
L-alanine emerged at 32 min and PITC-ꢀ-alanine emerged at
28 min. The concentrations of product formed in quenched
samples were determined from the fractions of the PITC
derivatives in the HPLC analysis. The peak area of either PITC-
L-alanine or PITC-ꢀ-alanine was divided by the sum of the two
and multiplied by the starting concentration of either L-alanine
or ꢀ-alanine to obtain molar concentrations. The starting
concentrations of L-alanine and ꢀ-alanine in quenched samples
were measured by reference to amino acid standards.
In the kinetic analysis with L-alanine as the substrate or
ethylamine as the activator, the standard procedure was followed
by the following modifications. Initial rates were determined at
varying substrate concentrations from 0.012 to 0.265 M L-
alanine or at 0.2 M L-alanine with varying ethylamine concen-
trations from 0.012 to 0.20 M. The initial rates were employed
in the calculation of kinetic parameters by the nonlinear least-
squares curve fitting programs (hypero and sigmoid) of Cle-
land.16
In assays, components were substituted as appropriate: 0.2
M methylamine or propylamine for ethylamine; 0.2 M L-2-
aminobutyrate for L-alanine; and 0.2 M sodium formate, sodium
acetate, or sodium bicarbonate with L-alanine in trials with
glutamate 2,3-aminomutase. The PITC derivatives of L-2-
aminobutyrate and DL-3 aminobutyrate (25-50 µL) were
separated as described above, except the gradient was modified
to 0-10% B in 40 min, 10-35% B in 35 min, and 35-100%
B in 15 min. PITC-L-2-aminobutyrate emerged at 68 min and
PITC-DL-3-aminobutyrate emerged at 58 min.
Assays of the primary kinetic isotope effect were conducted
at 190 mM of either L-alanine or L-[2,3,3,3-2H4]alanine with
200 mM ethylamine other assay conditions, as described above.
The concentration of each amino acid was established prior to
assay by quantitative amino acid analysis of PITC derivatives
referenced to L-alanine standards.
EPR Spectroscopy. Samples of LAM (450 µM subunits)
reductively incubated with or without 0.25 mM L-lysine were
quickly mixed anaerobically at 24 °C with 240 mM Tris-sulfate,
1.4 mM SAM, 1.5 mM sodium hydrosulfite, and 120 mM
L-alanine. In two experiments, 0.2 M ethylamine or propylamine
was also present as activators, and in one experiment, the
alkylamine activator was excluded. A control sample containing
120 mM L-lysine without L-alanine was prepared as above. The
samples were frozen at -150 °C as soon as possible after mixing
(<30 s). The EPR spectra of the frozen samples were recorded
at 77 K using a Varian model E3 spectrometer with the
following settings: field center, 3250 G; scan width, 200 G;
microwave frequency, 9.1 GHz; microwave power, 5 mW;
modulation frequency, 100 kHz; modulation amplitude, 1.6 G;
time constant, 0.3 s; scan time, 240 s; gain, 125 000.
2,3-Aminomutase and Glutamate 2,3-Aminomutasea
enzyme
substrate
L-alanine
activator
specific activity
lysine 2,3-
aminomutase
+ ethylamine
12 ( 0.34
- ethylamine
+ methylamine
0.21 ( 0.02
5.7 ( 0.16
+ n-propylamine
+ ethylamine
0.32 ( 0.03
1.2 ( 0.13
140 ( 4.2
3.7 ( 0.11
ndb
ꢀ-alanine
L-2-aminobutyrate + methylamine
- methylamine
glutamate 2,3-
aminomutase
L-alanine
+ formate
+ acetate
+ bicarbonate
nd
nd
a Activities were measured at 37 °C at pH 8.0 (NaEPPS) with
0.16 mM SAM, 0.7 mM Na-hydrosulfite, and 85 µM LAM
(subunits) in the anaerobic chamber. Substrate and amine or
carboxylate concentrations were 0.2 M. Reactions were stopped at
timed intervals, and products were derivatized as phenyiso-
thiocyanates (PITCs). The PITC derivatives were measured
spectrophotometrically after separation by HPLC. Specific activities
are expressed as nmol min-1 mg enzyme-1 b nd: not detectable
.
(activity < 0.1 nmol min-1 mg enzyme-1).
TABLE 2: Kinetic Parameters in the Action of Lysine
2,3-Aminomutasea
parameter
KEA (mM)
value
46 ( 6b
L-alanine (0.265 M) +
+
varied C2H5NH3
L-alanine (varied) +
KS (mM)
k (M-1 s-1
kH/kD
29 ( 4c
+
0.2 M C2H5NH3
L-alanine (varied) +
)
)
0.032 ( 0.002c
7.1
+
0.2 M C2H5NH3
L-alanine/[2,3-2H4+]alanine +
0.2 M C2H5NH3
L-alanine (varied) +
k (M-1 s-1
0.040 ( 0.002d
8.0 × 103
+
saturation M C2H5NH3
e
L-lysine (varied)
kcat/Km (s-1 M-1
)
a pH 8.0, 37 °C, 0.2 M Na-EPPS buffer, 0.16 mM SAM, 85 µM
LAM subunits. b Primary data in Figure S1 of Supporting
Information. c Apparent value by fitting eq 5 to data in Figure 1.
d Value at saturation of C2H5NH3 obtained by correction for 81%
+
saturation at 0.20 M. e Literature value of apparent second order rate
constant at pH 8.0 and 37 °C.9
activation by ethylamine displays saturation with respect to
ethylamine and obeys the Michaelis-Menten equation {V )
V[EA]/([EA] + KEA}, where V is the measured velocity at a
given concentration of ethylamine (EA), V is the maximum
velocity at saturating ethylamime, and KEA is the dissociation
constant of ethylamine. The value of KEA at 0.265 M L-alanine
is 46 mM (Table 2). Ethylamine at 0.2 M accelerates the
transformation of 0.2 M L-alanine into ꢀ-alanine 57-fold
(Table 1). At 0.2 M, ethylamine is at 81% saturation, so that
the acceleration would be 70-fold at saturating ethylamine.
Methylamine at 0.2 M also accelerates the reaction of
L-alanine 27-fold (Table 1). n-Propylamine displays very little
stimulation in the reaction of L-alanine (Table 1).
By the principle of microscopic reversibility, LAM must
catalyze the transformation of ꢀ-alanine into L-alanine. LAM
does catalyze the reverse reaction at about one-tenth the rate of
the forward reaction under comparable conditions (Table 1).
L-2-Aminobutyrate at 0.2 M reacts 18 times more rapidly than
0.2 M L-alanine (Table 1). Methylamine at 0.2 M stimulates
the reaction of L-2-aminobutyrate 38-fold.
Results and Discussion
Activation of Reaction of L-Alanine by Primary Amines.
LAM is highly selective for L-lysine as a substrate and does
not display detectable activity toward L-glutamate, L-glutamine,
L-threonine, L-homoserine, L-tyrosine, L-phenylalanine, L-valine,
L-leucine, L-isoleucine, L-histidine, L-tryptophan, L-ornithine,
or L-arginine. LAM displays very low but measurable activity
toward 0.2 M L-alanine and 0.2 M L-2-aminobutyrate, producing
ꢀ-alanine and L-3-aminobutyrate, respectively (Table 1). The
rates in Table 1 for 0.2 M L-alanine or L-2-aminobutyrate are,
respectively, 5 × 10-6 and 8 × 10-5 times the rate with
saturating L-lysine.
The primary amines presumably stimulate reactions of
L-alanine and L-2-aminobutyrate by binding to the active site
in place of the propylamine moiety in the L-lysyl side chain,
The presence of ethylamine significantly enhances the
action of LAM on L-alanine (Table 1). The kinetics of