Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.
A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Waldrop
G L
GL
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Turnbull
J L
JL
Parmentier
L E
LE
O'Leary
M H
MH
Cleland
W W
WW
Schachman
H K
HK
D000409:Alanine / Q000737:chemistry; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes; D002384:Catalysis; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013386:Succinates / Q000378:metabolism
D002247:Carbon Isotopes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013386:Succinates; D001224:Aspartic Acid; D006639:Histidine; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid; D000409:Alanine