A novel dye-linked formaldehyde dehydrogenase with some properties indicating the presence of a protein-bound redox-active quinone cofactor.
Dye-linked formaldehyde dehydrogenase from methylamine-grown Hyphomicrobium zavarzinii ZV 580, a tetramer of M(r) 210,000 with subunits of M(r) 54,000, was purified to homogeneity in five steps with 10% yield. The enzyme shows optimal affinity for, and activity with, formaldehyde (Km 67 microM) compared with other aldehydes. Pyridoxal phosphate, pyrroloquinoline quinone and other cofactors that would give the enzyme a distinctive absorption spectrum are absent. Slight changes are observed in the spectrum at 300-550 nm on oxidation of the enzyme with Wurster's Blue (WB) and reduction with formaldehyde. Titration of the native reduced enzyme with WB accounts for 2 mol of electrons per mol of tetrameric enzyme. The circumstantial evidence supporting the presence of a redox-active quinone cofactor bound to the polypeptide chain comprises a signal at g = 2.0049 in the X-band e.p.r. spectrum of the enzyme oxidized with WB, which disappears on reduction with formaldehyde, and a positive reaction of the native as well as the denatured and dialysed enzyme in the redox-cycling assay with glycinate and NitroBlue Tetrazolium (quinone staining). The oxidized enzyme is inhibited by equimolar amounts of phenylhydrazine, which is also a reductant. Hydrazone formation was absent with completely inhibited enzyme, according to photometric evidence. Likewise, the glycinate-dependent reduction of NitroBlue Tetrazolium was not affected by the inhibitor. It is concluded that an oxidation product of the hydrazine is the actual inhibitor which reacts with an amino acid residue of the active site rather than with the prospective quinone cofactor.
Klein
C R
CR
Botanisches Institut, Universität Bonn, Germany.
Kesseler
F P
FP
Perrei
C
C
Frank
J
J
Duine
J A
JA
Schwartz
A C
AC
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000447:Aldehydes; D001419:Bacteria / Q000201:enzymology; D003067:Coenzymes / Q000378:metabolism; D004396:Coloring Agents; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005557:Formaldehyde; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011809:Quinones / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D000447:Aldehydes; D003067:Coenzymes; D004396:Coloring Agents; D011809:Quinones; D005557:Formaldehyde; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent