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

Abstract

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity 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 imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the 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 still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing 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 base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

“…His-114 and His-115, the next upstream neighbors of loop 104 -113, are part of the active site. For His-114, an essential function in PDC catalysis has been proposed from kinetic studies with accordant variants from yeast and bacteria (7,8,42,43). Tittmann et al (44) postulated a specific role for His-114 (together with Asp-28) during release of the reaction product acetaldehyde.…”

“…The catalytic activity of PDC depends on the presence of the cofactor thiamine diphosphate (ThDP), which is bound mainly via a divalent metal ion (magnesium in most cases) to the protein moiety. Many detailed kinetic studies have been published on yeast PDC wild types (1)(2)(3)(4)(5)(6)(7)(8)(9). A number of ScPDC variants were analyzed, too (1)(2)(3)(4)(5)(6)(7)(8)(9).…”

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

“…Several regions were highly conserved, including the regions containing residues catalyzing the transition state intermediate during catalysis ( Fig. 2) (21) and residues involved in binding of the substrate and cofactors (31). The alignment in Fig.…”

“…base catalyst and Asp29, His112, His113, and Glu462 are involved in stabilization of the transition state intermediate (21,31).…”

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

“…Overexpression of the E477Q and D28A active site variants of YPDC was carried out following the protocol described previously [11]. The E477Q variant has a His 6 -tag attached to its C-terminal residue hence it was purified on a Talon column [11].…”