Consequences of a modified putative substrate-activation site on catalysis by yeast pyruvate decarboxylase.
Earlier, it had been proposed in the laboratories at Halle that a cysteine residue is responsible for the hysteretic substrate activation behavior of yeast pyruvate decarboxylase. More recently, this idea has received support in a series of studies from Rutgers with the identification of residue C221 as the site where substrate is bound to transmit the information to H92, to E91, to W412, and finally to the active center thiamin diphosphate. According to steady-state kinetic assays, the C221A/C222A variant is no longer subject to substrate activation yet is still a well-functioning enzyme. Several further experiments are reported on this variant: (1) The variant exhibits lag phases in the product formation progress curves, which can be attributed to a unimolecular step in the pre-steady-state stage of catalysis. (2) The rate of exchange with solvent deuterium of the thiamin diphosphate C2H atom is slowed by a factor of 2 compared to the wild-type enzyme, suggesting that the reduced activity that results from the substitutions some 20 A from the active center is also seen in the first key step of the reaction. (3) The solvent (deuterium oxide) kinetic isotope effect was found to be inverse on V(max)/K(m) (0.62), and small but normal on V(max) (1.26), virtually ruling out residue C221 as being responsible for the inverse effects reported for the wild-type enzyme at low substrate concentrations. The solvent kinetic isotope effects are compared to those on two related enzymes not subject to substrate activation, Zymomonas mobilis pyruvate decarboxylase and benzoylformate decarboxylase.
Wang
J
J
Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA.
Golbik
R
R
Seliger
B
B
Spinka
M
M
Tittmann
K
K
Hübner
G
G
Jordan
F
F
D000409:Alanine / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000235:genetics; D017666:Deuterium Oxide / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D011767:Pyruvate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000235:genetics; D012997:Solvents; D013379:Substrate Specificity / Q000235:genetics; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D011994:Recombinant Proteins; D012997:Solvents; D013844:Thiazoles; D012694:Serine; D006859:Hydrogen; D011767:Pyruvate Decarboxylase; D017666:Deuterium Oxide; D003545:Cysteine; D000409:Alanine; D013835:Thiamine Pyrophosphate