Interdomain Information Transfer during Substrate Activation of Yeast Pyruvate Decarboxylase: The Interaction between Cysteine 221 and Histidine 92†
journal contributionposted on 16.01.1998, 00:00 by Irina Baburina, Haijuan Li, Brian Bennion, William Furey, Frank Jordan
Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 22.214.171.124) from Saccharomyces cerevisiae at two cysteines on the β domain (221 and 222) and at H92 on the α domain, across the domain divide from C221. While C221 has been shown to provide the trigger for substrate activation [Baburina, I., et al. (1994) Biochemistry 33, 5630−5635], the information must be transmitted from the substrate bound at this site [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590−600] to the active center thiamin diphosphate located at the interface of the α and γ domains. Substitution at H92 with G, A, or C leads to great reduction of the Hill coefficient (from 2.0 in the wild-type enzyme to 1.2−1.3), while substitution for Lys affords an active enzyme with a Hill coefficient of 1.5−1.6. Iodoacetate at 10 mM reduced the Hill coefficient from 2.0 to 1.1, while also causing significant inactivation of the enzyme, presumably by carboxymethylation of C221. 1,3-Dibromoacetone, a potential cross-linker when added to the H92C/C222S variant at 0.1 mM, abolished substrate activation while reducing the activity only by 30%. Therefore, 1,3-dibromoacetone may cross-link C92 and C221. It was concluded that H92 is on the information transfer pathway during the substrate activation process and the interaction between C221 on the β domain and H92 on the α domain is required for substrate activation. Extensive pH studies of the steady-state kinetic constants provide support for the interaction of C221 and H92 and the transmission of regulatory information to the active center via this pathway and pKas for the two groups. This important interaction between the C221-bound pyruvate and His92 probably has both electrostatic and steric components.