HUMAN ALDOSE REDUCTASE - PK OF TYROSINE-48 REVEALS THE PREFERRED IONIZATION STATE FOR CATALYSIS AND INHIBITION

Detailed analyses of the pH variation of kinetic parameters for the fo rward aldehyde reduction and reverse alcohol oxidation reactions media ted by recombinant human aldose reductase, for inhibitor binding, and for kinetic isotope effects on aldehyde reduction have revealed that t he pK value for the active site acid-base catalyst group Tyr48 is quit e sensitive to the oxidation state of the bound nucleotide (NADPH or N ADP(+)) and to the presence or absence of the Cys298 sulfhydryl moiety . Thus, the Tyr48 residue of C298A mutant enzyme displays a pK value t hat ranges from 7.6 in the productive E . NADP(+) complex that binds and reacts with alcohols to 8.7 in the productive E . NADPH complex t hat binds and reacts with aldehyde substrates. For wild-type enzyme, T yr48 in the latter complex displays a lower pK value of about 8.25. As signment of the pK values was facilitated by the recognition and quant itation of the degree of stickiness of several aldehyde substrates in the forward reaction. The unusual pH dependence for V-aldehyde/E(t) an d V-D(aldehyde), which decrease roughly 20-fold through a wave and rem ain constant at high pH, respectively, is shown to arise from the pH-d ependent decrease in the net rate of NADP(+) release. The results desc ribed are fully consistent with the chemical mechanism for aldose redu ctase catalysis proposed previously (Bohren et al., 1994) and, further more, establish that binding of the spirohydantoin class of aldose red uctase inhibitors, e.g., sorbinil, occurs via a reverse protonation sc heme in which the ionized inhibitor binds preferentially to the E . N ADP(+) complex with Tyr48 present as the protonated hydroxyl form. The latter finding allows us to propose a unified model for high-affinity aldose reductase inhibitor binding that focuses on the transition sta te-like nature of the E-Tyr48-OH . NADP(+) . inhibitor(-)complex.