Bovine liver formaldehyde dehydrogenase. Kinetic and molecular properties.
The dimeric formaldehyde dehydrogenase from bovine liver has been resolved into three nearly homogeneous enzyme forms by the successive use of ion-exchange, affinity, and ampholine (chromatofocusing) chromatography. The different enzyme species were isolated in the approximate proportions 3:2:1, having pI values of 6.5, 6.2, and 6.0, respectively. The subunit molecular weights of the three forms are all similar (Mr congruent to 41,000), on the basis of sodium dodecyl sulfategel electrophoresis. The enzyme species appear to arise from covalent differences unrelated either to partial proteolysis during isolation or to differential sialization of homodimeric protein. Human liver contains a single major form and two minor forms of formaldehyde dehydrogenase having pI values very similar to those found for the bovine liver enzyme. The macroscopic kinetic constants (V, V/K) for the three forms of the dehydrogenase from bovine liver are all similar in magnitude, using NADH and S-hydroxymethylglutathione as substrates. The isotope-sensitive hydride transfer step is not significantly rate-limiting during catalysis by any of the forms, as evidenced by the near-unity primary deuterium isotope effects on both V and V/KS (for S-hydroxymethylglutathione); catalysis may be limited by the rate of dissociation of at least one (and possibly both) of the product molecules. In support of rate-limiting dissociation of NAD+ in the normal reaction, V increases by approximately 22-fold and isotope effects of approximately 1.4 are observed on both V and V/KS, using the coenzyme analog 3-acetylpyridine adenine dinucleotide. Product dissociation from the active site appears to be accelerated by the presence of dilute denaturing agents, perhaps indicative of a rate-limiting conformational transition associated with product release.
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Department of Chemistry, University of Maryland Baltimore County 21228.
Shih
M J
MJ
Creighton
D J
DJ
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003903:Deuterium; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011489:Protein Denaturation; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D007527:Isoenzymes; D009243:NAD; D014508:Urea; D003903:Deuterium; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases; D019791:Guanidine