The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.
Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(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
Lee
S
S
O'Leary
M H
MH
Cleland
W W
WW
Schachman
H K
HK
D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002221:Carbamyl Phosphate / Q000378:metabolism; D002247:Carbon Isotopes; D002384:Catalysis; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D009841:Oligonucleotides / Q000737:chemistry; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013386:Succinates / Q000378:metabolism; D013912:Threonine / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D013386:Succinates; D013912:Threonine; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D003903:Deuterium; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid