Role of glutamate 91 in information transfer during substrate activation of yeast pyruvate decarboxylase

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruv ate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at E91, locate d on the putative substrate activation pathway and linking the alpha and ga mma domains of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Bioche mistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10 255], that information, via the substrate bound at C221, is transmitted to H92 on the a domain, across the domain divide from C221 [Baburina, I., et a l. (1998) Biochemistry 37, 1235-1244], thence to E91 on the a domain, and t hen on to W412 on the gamma domain [Li, H., and Jordan, F. (1999) Biochemis try 38, 10004-10012] and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mel. Biol. 256, 590-600]. Substitution at E91 with Q, D, or A led to modes t reductions in the specific activity (4-, 5-, and 30-fold), as well as in both the turnover number and the catalytic efficiency, in that order. Inter estingly, the Hill coefficient was only slightly reduced for the E91D varia nt, but cooperativity was virtually abolished for the E91Q and E91A variant s. The thermal stability of the variants was reduced in the following order : wild type > E91Q > E91D > E91A; circular dichroism and fluorescence exper iments also demonstrated that the tertiary structure of the enzyme was affe cted by these substitutions. The variants could be purified as apoenzymes, demonstrating their impaired ability to bind thiamin diphosphate. Apparentl y, the charge at residue 91 is quite important for maintaining optimal coop erativity. To maintain strong domain-domain interactions, the length of the side chain at position 91 with hydrogen bending potential to W412 is suffi cient.