Monovalent cation activation in Escherichia coli inosine 5 '-monophosphatedehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of i nosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the conco mitant reduction of NAD to NADH, Escherichia coli IMPDH is activated by K+, Rb+, NH4+, and Cs+.K+ activation is inhibited by Li+, Na+, Ca2+, and Mg2+. This inhibition is competitive versus K+ at high K+ concentrations, noncom petitive versus IMP, and competitive versus NAD, Thus monovalent cation act ivation is linked to the NAD site. K+ increases the rate constant for the p re-steady-state burst of NADH production, possibly by increasing the affini ty of NAD, Three mutant IMPDHs have been identified which increase the valu e of K-m for K+:Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type , both Asp13Ala and Glu469Ala are activated by all cations tested. Thus the se mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K+ binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K+ activation of Asp50Ala is inhibited by Li+, Na+, Ca2+, and Mg2+. However, this inhibition is non-competitive with respect to K+ a nd competitive with respect to both IMP and NAD, Asp50 interacts with resid ues that form a rigid wall in the IMP site; disruption of this wall would b e expected to decrease IMP binding, and the defect could propagate to the p roposed K+ site. Alternatively, this mutation could uncover a second monova lent cation binding site. (C) 2000 Academic Press.