Mutational analysis of substrate interactions with the active site of dialkylglycine decarboxylase.
Pyridoxal phosphate (PLP)-dependent enzymes catalyze many different types of reactions at the alpha-, beta-, and gamma-carbons of amine and amino acid substrates. Dialkylglycine decarboxylase (DGD) is an unusual PLP-dependent enzyme that catalyzes two reaction types, decarboxylation and transamination, in the same active site. A structurally based, functional model has been proposed for the DGD active site, which maintains that R406 is important in determining substrate specificity through interactions with the substrate carboxylate while W138 provides specificity for short-chain alkyl groups. The mechanistic roles of R406 and W138 were investigated using site-directed mutagenesis, alternate substrates, and analysis of steady-state and half-reaction kinetics. Experiments with the R406M and R406K mutants confirm the importance of R406 in substrate binding. Surprisingly, this work also shows that the positive charge of R406 facilitates catalysis of decarboxylation. The W138F mutant demonstrates that W138 indeed acts to limit the size of the subsite C binding pocket, determining specificity for 2,2-dialkylglycines with small side chains as predicted by the model. Finally, work with the double mutant W138F/M141R shows that these mutations expand substrate specificity to include l-glutamate and lead to an increase in specificity for l-glutamate over 2-aminoisobutyrate of approximately 8 orders of magnitude compared to that of wild-type DGD.
Fogle
Emily J
EJ
Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA 93407, USA.
Toney
Michael D
MD
D000621:Aminoisobutyric Acids / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D003653:Decarboxylation; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics
D000621:Aminoisobutyric Acids; D018698:Glutamic Acid; D002262:Carboxy-Lyases; C063519:2,2-dialkylglycine decarboxylase