Studies on pig muscle aldose reductase. Kinetic mechanism and evidence for a slow conformational change upon coenzyme binding.
Steady state kinetic analysis at pH 7.0 of the reduction of DL-glyceraldehyde by pig muscle aldose reductase showed that the enzyme follows a sequential ordered mechanism with NADPH binding first. However, the "off constant" for NADP+ in the forward direction was 1 order of magnitude less than the kcat. Analysis of this anomaly by pre-steady state kinetics using stopped-flow fluorescence spectroscopy showed that this could be accounted for by isomerization of the enzyme-NADP+ complex and that the rate of isomerization is the rate-limiting step. The rate constant for this step was of the same order of magnitude as the kcat for the forward reaction. Fluorescence emission spectra of free and NADP(H)-bound enzyme suggested a conformational change upon binding of coenzyme. In the reverse direction (oxidation of glycerol) pre-steady state and steady state kinetic analyses were consistent with the rate-limiting step occurring before isomerization of the enzyme-NADPH complex. We conclude, therefore, that during the kinetic mechanism of the reduction of aldehydes by aldose reductase, a slow (kinetically detectable) conformational change in the enzyme occurs upon coenzyme binding. Since NADPH and NADP+ bind to the enzyme very tightly, this has implications for the targeting and binding of drugs that are aldose reductase inhibitors.
Kubiseski
T J
TJ
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Hyndman
D J
DJ
Morjana
N A
NA
Flynn
T G
TG
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D003067:Coenzymes / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D003067:Coenzymes; D009249:NADP; D000449:Aldehyde Reductase; D005990:Glycerol