Frederick P. Schwarz

The role of Cys-298 in aldose reductase function.

Diabetic tissues are enriched in an “activated” form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP+ remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP+ to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP+ binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

ATP Hydrolysis and DNA Binding Confer Thermostability on the MCM Helicase

The minichromosome maintenance (MCM) helicase is the replicative helicase in archaea. The enzyme utilizes the energy derived from ATP hydrolysis to translocate along one strand of the DNA and unwind the complementary strand. Here, the effect of DNA and ATP on the thermostability of the Methanothermobacter thermautotrophicus MCM protein was determined by differential scanning calorimetry. The MCM protein shows a single thermal transition at 67 degrees C. The stability is dramatically altered with the appearance of a second thermal transition up to 10 degrees C higher in the presence of DNA and either ATP or ADP-AlF(4)(-), a transition-state analogue of ATP, bound to MCM. In the presence of DNA and ADP or the nonhydrolyzable ATP analogues ATPgammaS and AMP-PNP, however, only a single thermal transition is observed at temperatures slightly higher than the transition temperature of MCM alone. Thus, the results suggest that ATP hydrolysis proceeds through a transition state that decouples an interaction between the N-terminal DNA binding domain and the C-terminal catalytic domain in the presence of DNA.