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 forward aldehyde reduction and reverse alcohol oxidation reactions mediated by recombinant human aldose reductase, for inhibitor binding, and for kinetic isotope effects on aldehyde reduction have revealed that the pK value for the active site acid-base catalyst group Tyr48 is quite sensitive to the oxidation state of the bound nucleotide (NADPH or NADP+) and to the presence or absence of the Cys298 sulfhydryl moiety. Thus, the Tyr48 residue of C298A mutant enzyme displays a pK value that 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 that binds and reacts with aldehyde substrates. For wild-type enzyme, Tyr48 in the latter complex displays a lower pK value of about 8.25. Assignment of the pK values was facilitated by the recognition and quantitation of the degree of stickiness of several aldehyde substrates in the forward reaction. The unusual pH dependence for Valdehyde/Et and DValdehyde, which decrease roughly 20-fold through a wave and remain constant at high pH, respectively, is shown to arise from the pH-dependent decrease in the net rate of NADP+ release. The results described are fully consistent with the chemical mechanism for aldose reductase catalysis proposed previously (Bohren et al., 1994) and, furthermore, establish that binding of the spirohydantoin class of aldose reductase inhibitors, e.g., sorbinil, occurs via a reverse protonation scheme in which the ionized inhibitor binds preferentially to the *E.NADP+ 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 state-like nature of the *E-Tyr48-OH.NADP+.inhibitor- complex.
Grimshaw
C E
CE
Whittier Diabetes Program, Department of Medicine, University of California, San Diego, La Jolla 92093-0983, USA.
Bohren
K M
KM
Lai
C J
CJ
Gabbay
K H
KH
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D014443:Tyrosine / Q000737:chemistry
D004791:Enzyme Inhibitors; D014443:Tyrosine; D000449:Aldehyde Reductase