Differences in catalytic activity between rat testicular and ovarian carbonyl reductases are due to two amino acids.
The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K(m) and 5-fold lower k(cat) towards menadione and a 7-fold lower K(m) and 7-fold lower k(cat) towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K(m) for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.
Sciotti
Michel A
MA
Institute of Clinical Chemistry, 3010 Bern, Switzerland.
Tam
Steven
S
Wermuth
Bendicht
B
Baker
Michael E
ME
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D005260:Female; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D009928:Organ Specificity / Q000502:physiology; D010053:Ovary / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D040681:Structural Homology, Protein; D013737:Testis / Q000201:enzymology*; D024483:Vitamin K 3 / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D011725:Pyridines; C066738:4-benzoylpyridine; D024483:Vitamin K 3; D000429:Alcohol Oxidoreductases