Substrate- and inhibitor-induced conformational changes in enzymes measured by tritium-hydrogen exchange. II. Yeast pyruvate decarboxylase.

The effects of substrates and inhibitors on the conformation of yeast cytoplasmic pyruvate decarboxylase (Z-oxo-acid carboxy-lyase, EC 4 .l .l .l) has been examined using the technique of automated tritium-hydrogen exchange. This experimental approach, as used in this study, reflects primarily the number of solvent-inaccessible or “core” hydrogens in the protein. Yeast cytoplasmic pyruvate decarboxylase is a multisubunit enzyme, molecular weight 208,000, and contains thiamine pyrophosphate as a co-factor at the active site. We find that 850 out of a total of 1600 amide hydrogens are measurable by tritium exchange at pH 6.5, 0”. There are 225 (or 14% of the total) very slowly exchanging hydrogens which we believe arise either from solvent-inaccessible regions of the subunits or from interface areas between the subunits. Upon addition of substrate, pyruvate, inhibitor, cy-ketoglutarate, or mercuric chloride, a marked conformational change occurs in which 50% of the very slowly exchanging hydrogens become rapidly exchanging. Lactate and formaldehyde have no effect. Mercuric chloride causes a partial conformational change, but the enzyme is still capable of binding pyruvate and exhibiting a further release of slowly exchanging hydrogens. The effects of pyruvate and mercuric chloride are not additive; therefore, we believe that they are acting at common areas on the enzyme. The enzyme is not dissociated into subunits by the substrate. Thus, this enzyme has a conformationally regulated accessibility to its active site. It is proposed that the conformational changes we observe may be very similar to those obtained during allosteric modification of enzymes and may involve a reorganization of subunit interfaces.