Solvent kinetic isotope effects monitor changes in hydrogen bonding at the active center of yeast pyruvate decarboxylase concomitant with substrate activation: the substituent at position 221 can control the state of activation.
Substrate activation of yeast pyruvate decarboxylase has been studied extensively in the authors' laboratories providing strong evidence that interaction of substrate with residue C221 provides the trigger, and the information is then transmitted along the C221 to H92 to E91 to W412 to G413 pathway to the 4'-amino nitrogen of the thiamin diphosphate cofactor. Earlier, it was found that the C221S substitution reduced the Hill coefficient from 2.0 to 0.8-0.9, the C221A substitution to 1.0, even though C221 is located on the beta domain some 20 A from the active center thiamin diphosphate cofactor, which is at the interface of the alpha and gamma domains. Here are reported experiments on the C221D/C222A and C221E/C222A variants, in which a negative charge is built onto the C221 side chain, to better mimic the effect of a pyruvate molecule covalently bonded to C221 as a thiohemiketal. Both variants were purified to an optimal activity of 70% of the wild-type enzyme, higher activity than that with the earlier uncharged substitutions at this position. The Hill coefficient for both variants is exactly 1.0. The deuterium solvent kinetic isotope effects (SKIE) on k(cat) for these variants were similar to that for the wild-type enzyme and the C221A/C222A variant, suggesting that starting with the first irreversible step (decarboxylation) the rate-limiting transition states are very similar for all of these enzyme forms. In contrast, such SKIE on k(cat)/K(m) are quite different for the C221A/C222A variant (0.62) than for the C221E/C222A or C221D/C222A variants (0.80-0.82), clearly indicating the effect of the C221 substitutions on transition states starting with the binding of the first substrate to the enzyme and terminating with the decarboxylation step. The results provide strong additional evidence for the involvement of residue C221 in the substrate activation process and suggest that the C221D (C221E) substitution shifts the enzyme into a conformation that resembles the activated conformation. A comparison with SKIE for the wild-type enzyme provides insight to changes in hydrogen bonding at the active center as a result of substrate activation.
Wei
Wen
W
Department of Chemistry and Program in Cellular and Molecular Biodynamics, Rutgers, the State University of New Jersey, Newark, New Jersey 07102, USA.
Liu
Min
M
Jordan
Frank
F
D001665:Binding Sites; D004789:Enzyme Activation; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011767:Pyruvate Decarboxylase / Q000737:chemistry*; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry; D017355:Zymomonas / Q000201:enzymology*
D014867:Water; D011767:Pyruvate Decarboxylase