Studies on the active site of pig kidney aldehyde reductase

1981 ◽  
Vol 9 (4) ◽  
pp. 273-275 ◽  
Author(s):  
T. GEOFFREY FLYNN ◽  
CHRISTINE GALLERNEAULT ◽  
DAVID FERGUSON ◽  
JAMES A. CROMLISH ◽  
WILLIAM S. DAVIDSON
Keyword(s):  
Active Site ◽  
Aldehyde Reductase ◽  
Pig Kidney

scholarly journals Some properties of pig kidney-cortex aldehyde reductase

1980 ◽  
Vol 191 (2) ◽  
pp. 619-626 ◽  
Author(s):  
F F Morpeth ◽  
F M Dickinson
Keyword(s):  
Active Site ◽  
Fluorescence Spectra ◽  
Dissociation Constants ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Kidney Cortex ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Binary Complexes ◽  
Spectrophotometric Titrations

Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.

scholarly journals Evidence for the importance of arginine residues in pig kidney alkaline phosphatase

1979 ◽  
Vol 181 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M N Woodroofe ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Close Proximity ◽  
Arginine Residues ◽  
Km Value ◽  
Uncompetitive Inhibitor ◽  
Kidney Alkaline Phosphatase

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

Mechanism of Human Aldehyde Reductase: Characterization of the Active Site Pocket

Biochemistry ◽  
1995 ◽  
Vol 34 (35) ◽  
pp. 11264-11275 ◽  
Author(s):  
Oleg A. Barski ◽  
Kenneth H. Gabbay ◽  
Charles E. Grimshaw ◽  
Kurt M. Bohren
Keyword(s):  
Active Site ◽  
Aldehyde Reductase ◽  
Active Site Pocket

scholarly journals Kinetic studies of the mechanism of pig kidney aldehyde reductase

1981 ◽  
Vol 193 (2) ◽  
pp. 485-492 ◽  
Author(s):  
F F Morpeth ◽  
F M Dickinson
Keyword(s):  
Initial Rate ◽  
Kinetic Studies ◽  
Random Order ◽  
Alcohol Oxidation ◽  
Aldehyde Reductase ◽  
Pig Kidney ◽  
Binary Complexes ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Aldehyde Reduction

Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.

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