Itwas concluded that H92 is on the information transfer pathway during the substrate activation processand the interaction between C221 on the domain and H92 on theR domain is required for substrateactivation. Therefore, 1,3-dibromoacetone may cross-link C92 and C221. 1,3-Dibromoacetone, a potentialcross-linker when added to the H92C/C222S variant at 0.1 mM, abolished substrate activation whilereducing the activity only by 30%. Iodoacetate at 10 mM reduced the Hill coefficient from 2.0 to 1.1, while also causing significantinactivation of the enzyme, presumably by carboxymethylation of C221. While C221 has been shown to provide thetrigger for substrate activation, the informationmust be transmitted from the substrate bound at this site to the active center thiamin diphosphate located at the interface of theR and domains.Substitution at H92 with G, A, or C leads to great reduction of the Hill coefficient (from 2.0 in thewild-type enzyme to 1.2-1.3), while substitution for Lys affords an active enzyme with a Hill coefficientof 1.5-1.6. ReceiVed ApReVised Manuscript ReceiVed September 24, 1997ĪBSTRACT: Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase(EC 4.1.1.1) fromSaccharomyces cereVisiaeat two cysteines on the domain (221 and 222) and at H92on theR domain, across the domain divide from C221. Irina Baburina, Haijuan Li, Brian Bennion, William Furey, and Frank Jordan*,ĭepartment of Chemistry and Program in Cellular and Molecular Biodynamics, Rutgers, the State UniVersity of New Jersey,73 Warren Street, Newark, New Jersey 07102, and Department of Crystallography, VA Medical Center,
Interdomain Information Transfer during Substrate Activation of Yeast PyruvateDecarboxylase: The Interaction between Cysteine 221 and Histidine 92
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