Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.