MECHANISTIC STUDIES ON MALATE-DEHYDROGENASE FROM ESCHERICHIA-COLI

Kinetic studies and chemical modification studies using diethylpyrocar bonate and iodoacetate were performed on malate dehydrogenase isolated from Escherichia coli. Product inhibition experiments indicate that t his enzyme follows an ordered Bi Bi kinetic mechanism, similar to othe r dehydrogenases, while log V/K profiles reveal that one ionizing grou p with a pK(a) between 7.8 and 8.7 acts as a general acid/general base in the catalytic mechanism. Log V pro files indicate that malate bind s to the correctly protonated form of the enzyme while binding of OAA is pH-independent. Chemical modification experiments implicate an acti vate site histidine residue is essential for catalytical activity, A p rimary kinetic isotope effect of 1.44 +/- 0.14 on V/K using malate-2-d at pH 9.0 was measured while no isotope effect was observed on V-max which is, again, similar to other dehydrogenases, This implies that pr oton abstraction is partially rate determining under nonsaturating con ditions. Within experimental error, small isotope effects were observe d on V and V/K (1.219 +/-: 0.188 and 1.078 +/- 0.064, respectively) wh en NADD was utilized indicating that release of cofactor may be rate l imiting. (C) 1995 Academic Press, Inc.