Catalytic acid-base groups in yeast pyruvate decarboxylase. 3. A steady-state kinetic model consistent with the behavior of both wild-type and variant enzymes at all relevant pH values

The widely quoted kinetic model for the mechanism of yeast pyruvate decarbo xylase (YPDC, EC 4.1.1.1), an enzyme subject to substrate activation, is ba sed on data for the wild-type enzyme under optimal experimental conditions. The major feature of the model is the obligatory binding of substrate in t he regulatory site prior to substrate binding at the catalytic site. The ac tivated monomer would complete the cycle by irreversible decarboxylation of the substrate and product (acetaldehyde) release. Our recent kinetic studi es of YPDC variants substituted at positions D28 and E477 at the active cen ter necessitate some modification of the mechanism. It was found that enzym e without substrate activation apparently is still catalytically competent. Further, substrate-dependent inhibition of D28-substituted variants leads to an enzyme form with nonzero activity at full saturation, requiring a sec ond major branch point in the mechanism. Kinetic data for the E477Q variant suggest that three consecutive substrate binding steps may be needed to re lease product acetaldehyde, unlikely if YPDC monomer is the minimal catalyt ic unit with only two binding sites for substrate. A model to account for a ll kinetic observations involves a functional dimer operating through alter nation of active sites. In the context of this mechanism, roles are suggest ed for the active center acid-base groups D28, E477, H114, and H115. The re sults underline once more the enormous importance that both aromatic rings of the thiamin diphosphate, rather than only the thiazolium ring, have in c atalysis, a fact little appreciated prior to the availability of the 3-dime nsional structure of these enzymes.