Spectrophotometric and steady-state kinetic analysis of the biosynthetic arginine decarboxylase of Yersinia pestis utilizing arginine analogues as inhibitors and alternative substrates.
The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.
Balbo
Paul B
PB
College of Pharmacy, Center for Structural Biology, and Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536, USA.
Patel
Chandra N
CN
Sell
Korie G
KG
Adcock
Robert S
RS
Neelakantan
Sundar
S
Crooks
Peter A
PA
Oliveira
Marcos A
MA
D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry*; D002172:Canavanine / Q000737:chemistry; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000737:chemistry; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D015010:Yersinia pestis / Q000201:enzymology*; D019323:omega-N-Methylarginine / Q000737:chemistry
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019323:omega-N-Methylarginine; D002172:Canavanine; C000225:argininic acid; D001120:Arginine; D002262:Carboxy-Lyases; C019722:arginine decarboxylase