Mutants of 4-oxalocrotonate tautomerase catalyze the decarboxylation of oxaloacetate through an imine mechanism.
A designed single amino acid substitution can alter the catalytic activity and mechanism of 4-oxalocrotonate tautomerase (4-OT). While the wild-type enzyme catalyzes only the tautomerization of oxalocrotonate, the Pro1Ala mutant (P1A) catalyzes two reactions--the original tautomerization reaction and the decarboxylation of oxaloacetate. Although the N-terminal amine group of P1A is involved in both reactions, our results support a nucleophilic mechanism for the decarboxylase activity, in contrast to the general acid/base mechanism that has been previously established for the tautomerase activity. These findings demonstrate that a single catalytic group in a 4-OT mutant can catalyze two reactions by two different mechanisms.
Brik
Ashraf
A
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
D'Souza
Lawrence J
LJ
Keinan
Ehud
E
Grynszpan
Flavio
F
Dawson
Philip E
PE
D019943:Amino Acid Substitution; D002384:Catalysis; D003653:Decarboxylation; D007097:Imines / Q000737:chemistry; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D062907:Oxaloacetic Acid / Q000378:metabolism*; D015202:Protein Engineering; D016958:Pseudomonas putida / Q000201:enzymology
D007097:Imines; D062907:Oxaloacetic Acid; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases