5790 J. Am. Chem. Soc., Vol. 119, No. 25, 1997
Boehlein et al.
added, the mixture was gently heated until solvent reflux was observed.
Heating was then stopped, and the reaction was allowed to proceed at
ambient temperature for 1 h. The suspension was filtered through
Celite, which was subsequently washed with water. The resulting
solution was then lyophilized to yield 1 as a white solid, which was
recrystallized from dioxane/water: 1.2 g, 76%; mp 227-229 °C [lit.17a
227.5-229 °C]; [R]22 +38.8° (c ) 1.28, 1 M AcOH) [lit.17b [R]21
+32.2° (c ) 0.54, 1 M AcOH)]; IR (KBr) ν 2959, 2122, 1618, 1588,
1414, 1342, 1314, 1210, 1150, 1085, 1011 cm-1; 1H NMR (D2O, 500
MHz) δ 2.17-2.29 (2 H, dq, J ) 7.2, 10.6 Hz), 2.69 (2 H, dt, J ) 4.3,
7.5 Hz), 3.79 (1 H, t, J ) 6.7 Hz); 13C NMR (D2O, 75.4 MHz) δ 13.24
(t), 26.00 (t), 53.04 (d), 119.79 (s), 172.74 (s); HRMS (CI H+) exact
mass calcd for C5H9N2O2 requires 129.066, found 129.063; m/e (rel
intensity) 129 (100), 83 (42). Anal. Calcd for (C5H8N2O2): C, 46.87;
H, 6.29; N, 21.86. Found: C, 46.85; H, 6.45; N, 21.70.
The results outlined here raise the question of whether the
class II amidotransferases and thiol proteases are related by
common ancestry. Sequence analysis does not, however, appear
to support such a hypothesis for these two protein families, and
the three-dimensional folds of papain and the N-terminal
glutamine-utilizing domain of glutamine PRPP (5′-phosphori-
bosylpyrophosphate) amidotransferase are clearly different.
Therefore, it seems likely that the active sites of these two
families of enzymes has arisen through convergent evolution
and that the functional equivalence of Gln-19 in papain and
Asn-74 in AS-B is dictated by the chemical nature of the
transition states and intermediates that are formed during thiol-
catalyzed amide hydrolysis. Our studies provide further evi-
dence for the hypothesis that chemistry places strict limits on
the nature of catalytic residues in enzyme active sites and are
consistent with recent observations concerning members of the
enolase superfamily, in which anions are formed adjacent to
carboxylic moieties using chemically equivalent residues.29
Finally, our ability to reengineer the catalytic activity of the
N-terminal AS-B domain by mutation of a critical active site
residue, in a similar manner to that reported for papain,8 suggests
a strategy by which the modification of a single residue might
be employed to obtain enzymes with altered activity. Hence,
if an intermediate in the mechanism underlying WT activity
can be identified that is common to an alternate pathway, then
replacement of the protein side chain can be used to bias
partitioning of the intermediate to introduce a new activity. In
the example reported here, mutagenesis of Asn-74 affects the
tetrahedral intermediate that is common to the mechanisms of
nitrile hydration and amide hydrolysis. It is possible that such
an evolutionary mechanism has been operative in generating
the mechanistic diversity observed in the enzymes of the enolase
superfamily.
D
D
Expression and Purification of WT AS-B and the N74A and
N74D AS-B Mutants. Full experimental details of the construction,
expression, and purification of the proteins used in these studies have
been published previously.11b,14
Kinetic Characterization of the Glutaminase Activities of WT
AS-B and the N74A and N74D AS-B Mutants. (a) The glutaminase
activities of WT AS-B and the N74A AS-B mutant were assayed using
a spectroscopic procedure31 as previously described14 except that all
reactions were carried out using 100 mM Bis-Tris/100 mM Tris-HCl
as buffer. (b) Glutamine was incubated at 37 °C with 8 mM MgCl2,
and the N74D AS-B mutant (80 µg) was incubated at a concentration
of 2.5 mM in 100 mM Bis-Tris/100 mM Tris-HCl adjusted to pH 6.5.
The total reaction volume was 150 µL. At various times (0, 45, 90,
135, and 180 min), an aliquot (25 µL) of the mixture was taken and
reaction terminated by boiling. Samples were filtered through a 2 µm
filter and derivatized with phenylisothiocyanate (PITC). The glutamine
and glutamate derivatives were separated by HPLC and quantitated
spectroscopically. This assay procedure can detect less than 100 pmol
of product under standard conditions.
Enzyme Inhibition Assays. (a) The ability of wild type AS-B to
employ 1 as a substrate in asparagine synthesis was assayed by
incubating 10 mM nitrile 1 with 5.6 µg of enzyme, 10 mM aspartate,
5 mM ATP, and 8 mM MgCl2 in 100 mM HEPES buffer at pH 7.0
(100 µL total reaction volume) for 15 min at 37 °C. After the reaction
was terminated by boiling, no pyrophosphate production could be
detected in a coupled assay system (Sigma Technical bulletin number
B1-100), indicating that 1 was not a substrate for the synthetase activity
of enzyme. (b) The inhibition of the glutaminase activity of WT AS-B
by 1 was determined by measuring glutamate production using the
reaction of glutamate dehydrogenease in the presence of NAD+.31,14
Standard curves were obtained in the presence and absence of nitrile 1
and showed that the nitrile did not affect the activity of glutamate
dehydrogenase. In these studies, WT AS-B (1.5 µg) was incubated
for 15 min at 37 °C with 8 mM MgCl2 in HEPES buffer, pH 7 (total
volume 100 µL), at various glutamine concentrations (0.5, 0.75, 1.0,
1.5, 3.0, 5.0, and 7.5 mM). Nitrile concentrations of 0, 0.1, 0.3, and
0.5 mM were employed in determining the inhibition constants for 1.
Reaction mixtures were added to 380 µL of the coupling reagent (300
mM glycine, 250 mM hydrazine pH 9, 1 mM ADP, 1.6 mM NAD+,
and 2.2 U glutamate dehydrogenase) and incubated for 10 min at room
temperature. The solution absorbance was measured at 340 nm, and
the amount of glutamate present was determined from a standard curve.
All assays were performed in triplicate. In the inhibition studies at
pH 8, identical conditions were employed except that the buffer was
100 mM Tris-HCl and 2.5 µg of WT AS-B was present. (c) The
inhibition of the glutaminase activity of the N74D AS-B mutant by 1
was determined as described for the WT enzyme. In these studies, the
N74A AS-B mutant (14.7 µg) was incubated for 15 min at 37 °C with
8 mM MgCl2 in HEPES buffer, pH 7 (total volume 100 µL), at various
glutamine concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.75, and 1.0 mM).
Nitrile concentrations of 0, 0.1, 0.3, and 0.5 mM were employed in
determining the inhibition constants for 1. All assays were performed
in duplicate.
Experimental Section
Melting points were recorded using a Fisher-Johns melting point
apparatus and are uncorrected. Chemical shifts are reported in ppm
(δ) downfield of tetramethylsilane as an internal reference (δ 0.0).
Splitting patterns are abbreviated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet. Combustion analyses for C, H, and N
were determined in the Microanalysis Facility in the Department of
Chemistry, University of Florida. Analytical thin-layer chromatography
(TLC) was performed on silica gel 60F-245 plates, unless otherwise
stated. Flash chromatography was performed by standard methods30
on Davisil grade 633 type 60 Å silica gel (200-425 mesh). All solvents
were purified by distillation before use. Chemical compounds were
purchased from Aldrich and used without further purification.
2-((Carbobenzoxy)amino)-4-cyanobutyric Acid (3). This material
was prepared, from N-(carbobenzoxy)glutamine 2, using the literature
procedure,17a as a colorless oil in quantitative yield: IR (CHCl3) ν 3421,
3026, 2954, 2250, 1720, 1509, 1453, 1344, 1228, 1051 cm-1; 1H NMR
(CDCl3, 500 MHz) δ 1.99-2.08 (1 H, m), 2.20-2.32 (1 H, m), 2.39-
2.42 (2 H, m), 4.41-4.44 (1 H, m), 5.10 (2 H, s), 5.79 (1 H, d, J ) 7.8
Hz), 7.31-7.37 (5 H, m), 10.39 (1 H, br s); 13C NMR (CDCl3, 75.4
MHz) δ 13.53 (t), 27.93 (t), 52.55 (d), 67.39 (t), 118.71 (s), 128.06
(d), 128.29 (d), 128.50 (d), 135.65 (s), 156.24 (s), 173.94 (s); HRMS
(FAB H+) exact mass calcd for C13H15N2O4 requires 263.1032, found
263.1031; m/e (rel intensity) 263 (36), 91 (100).
2-Amino-4-cyanobutyric Acid (1). The protected nitrile 3 (1.33
g, 5.07 mmol) was dissolved in dry EtOH (10 mL) together with 1,4-
cyclohexadiene (2.4 mL). After 10% Pd/C (680 mg) as catalyst was
(29) (a) Babbitt, P. C.; Hasson, M. S.; Wedekind, J. E.; Palmer, D. R.
J.; Barrett, W. C.; Reed, G. H.; Rayment, I.; Ringe, D.; Kenyon, G. L.;
Gerlt, J. A. Biochemistry 1996, 35, 16489-16501. (b) Babbitt, P. C.;
Mrachko, G. T.; Hasson, M. S.; Huisman, G. W.; Kolter, R.; Ringe, D.;
Petsko, G. A.; Kenyon, G. L.; Gerlt, J. A. Science 1995, 267, 1159-1161.
(30) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-
2925.
Kinetic Characterization of the Nitrile Hydratase Activity of WT
AS-B and the N74D AS-B Mutant. (a) Nitrile 1 was incubated with
(31) Brent, E.; Bergmeyer, H. U. In Methods of Enzymatic Analysis;
Bergmeyer, H. U., Ed.; Academic Press: New York, 1974; pp 1704-1708.