Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases

Redox potentials for two inactivating intrasubunit disulfides that link hel ix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The E-m is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305, Oxidation to the disulfide affects k(cat) but not K-m values. In the single V121C and N1 22C mutants, the Cys in helix-Ei affects the K-m for oxalacetate, The pH op timum in the direction of malate formation is affected by the redox state o f the enzyme. Clearly, a disulfide bond can and does form between Cys resid ues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase, Apparently, crosslinking the domains interferes with cataly sis, (C) 2000 Academic Press.