Catalytic acid-base groups in yeast pyruvate decarboxylase. 1. Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions

The roles of four of the active center groups with potential acid-base prop erties in the region of pH optimum of pyruvate decarboxylase from Saccharom yces cerevisiae have been studied with the substitutions Asp28Ala, His114Ph e, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis method s. The steady-state kinetic constants were determined in the pH range of ac tivity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S-0.5 (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also i mply that all four are involved in some manner in stabilizing the rate-limi ting transition state(s) both at low substrate (steps starting with substra te binding and culminating in decarboxylation) and at high substrate concen tration (steps beginning with decarboxylation and culminating in product re lease). With the exception of some modest effects, the shapes of neither th e bell-shaped k(cat)/S-0.5-pH (and related functions) plots nor the k(cat)- pH plots are changed by the substitutions. Yet, the fractional activity sti ll remaining after substitutions virtually rules out any of the four residu es as being directly responsible for initiating the catalytic process by io nizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the ba se induced deprotonation at C2 is carried out by the only remaining base, t he iminopyrimidine tautomer of the coenzyme, via intramolecular proton abst raction. The first product is released as CO2 rather than HCO3- by both wil d-type and E477Q and D28A variants, ruling out several mechanistic alternat ives.