Ionization of zwitterionic amine substrates bound to monomeric sarcosine oxidase.
Monomeric sarcosine oxidase (MSOX) binds the L-proline zwitterion (pKa = 10.6). The reactive substrate anion is generated by ionization of the ES complex (pKa = 8.0). Tyr317 was mutated to Phe to determine whether this step might involve proton transfer to an active site base. The mutation does not eliminate the ionizable group in the ES complex (pKa = 8.9) but does cause a 20-fold decrease in the maximum rate of the reductive half-reaction. Kinetically determined Kd values for the ES complex formed with L-proline agree with results obtained in spectral titrations with the wild-type or mutant enzyme. Unlike the wild-type enzyme, Kd values with the mutant enzyme are pH-dependent, suggesting that the mutation has perturbed the pKa of a group that affects the Kd. As compared with the wild-type enzyme, an increase in charge transfer band energy is observed for mutant enzyme complexes with substrate analogues while a 10-fold decrease in the charge transfer band extinction coefficient is found for the complex with the L-proline anion. The results eliminate Tyr317 as a possible acceptor of the proton released upon substrate ionization. Since previous studies rule out the only other nearby base, we conclude that L-proline is the ionizable group in the ES complex and that amino acids are activated for oxidation upon binding to MSOX by stabilization of the reactive substrate anion. Tyr317 may play a role in substrate activation and optimizing binding, as judged by the effects of its mutation on the observed pKa, reaction rates, and charge transfer bands.
Zhao
Gouhua
G
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Jorns
Marilyn Schuman
MS
D000085:Acetates / Q000737:chemistry; D000465:Algorithms; D000588:Amines / Q000737:chemistry; D019943:Amino Acid Substitution; D000838:Anions / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011392:Proline / Q000031:analogs & derivatives / Q000737:chemistry*; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012521:Sarcosine / Q000737:chemistry; D050879:Sarcosine Oxidase / Q000737:chemistry* / Q000235:genetics; D013053:Spectrophotometry; D013379:Substrate Specificity; D017186:Titrimetry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D000085:Acetates; D000588:Amines; D000838:Anions; D004791:Enzyme Inhibitors; D011522:Protons; D011994:Recombinant Proteins; D014443:Tyrosine; C002986:2-pyrrolecarboxylic acid; D011392:Proline; D050879:Sarcosine Oxidase; C026500:methyl thioacetate; D012521:Sarcosine