Molecular basis for differential substrate specificity in class IV alcohol dehydrogenases -: A conserved function in retinoid metabolism but not in ethanol oxidation

被引:32
作者
Crosas, B
Allali-Hassani, A
Martínez, SE
Martras, S
Persson, B
Jörnvall, H
Parés, X [1 ]
Farrés, J
机构
[1] Univ Autonoma Barcelona, Dept Biochem & Mol Biol, E-08193 Barcelona, Spain
[2] Karolinska Inst, Dept Med Biochem & Biophys, S-17177 Stockholm, Sweden
关键词
D O I
10.1074/jbc.M910040199
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mammalian class IV alcohol dehydrogenase enzymes are characteristic of epithelial tissues, exhibit moderate to high K-m values for ethanol, and are very active in retinol oxidation, The human enzyme shows a K-m value for ethanol which is 2 orders of magnitude lower than that of rat class IV. The uniquely significant difference in the substrate-binding pocket between the two enzymes appears to be at position 294, Val in the human enzyme and Ala in the rat enzyme. Moreover, a deletion at position 117 (Gly in class I) has been pointed out as probably responsible for class IV specificity toward retinoids, With the aim of establishing the role of these residues, we have studied the kinetics of the recombinant human and rat wild-type enzymes, the human G117ins and V294A mutants, and the rat A294V mutant toward aliphatic alcohols and retinoids, 9-cis-Retinol was the best retinoid substrate for both human and rat class IV, strongly supporting a role of class IV in the generation of 9-cis-retinoic acid. In contrast, 13-cis retinoids were not substrates, The G117ins mutant showed a decreased catalytic efficiency toward retinoids and toward three-carbon and longer primary aliphatic alcohols, a behavior that resembles that of the human class I enzyme, which has Gly(117). Th, K-m values for ethanol dramatically changed in the 294 mutants, where the human V294A mutant showed a 280-fold increase, and the rat A294V mutant a 50-fold decrease, compared with those of the respective wild-type enzymes. This demonstrates that the Val/Ala exchange at position 294 is mostly responsible for the kinetic differences with ethanol between the human and rat class IV. In contrast, the kinetics toward retinoids was only slightly affected by the mutations at position 294, compatible with a more conserved function of mammalian class IV alcohol dehydrogenase in retinoid metabolism.
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页码:25180 / 25187
页数:8
相关论文
共 42 条
[1]   BIASED PROBABILITY MONTE-CARLO CONFORMATIONAL SEARCHES AND ELECTROSTATIC CALCULATIONS FOR PEPTIDES AND PROTEINS [J].
ABAGYAN, R ;
TOTROV, M .
JOURNAL OF MOLECULAR BIOLOGY, 1994, 235 (03) :983-1002
[2]   Retinoids, ω-hydroxyfatty acids and cytotoxic aldehydes as physiological substrates, and H2-receptor antagonists as pharmacological inhibitors, of human class IV alcohol dehydrogenase [J].
Allali-Hassani, A ;
Peralba, JM ;
Martras, S ;
Farrés, J ;
Parés, X .
FEBS LETTERS, 1998, 426 (03) :362-366
[3]   Alcohol dehydrogenase of human and rat blood vessels - Role in ethanol metabolism [J].
AllaliHassani, A ;
Martinez, SE ;
Peralba, JM ;
Vaglenova, J ;
Vidal, F ;
Richart, C ;
Farres, J ;
Pares, X .
FEBS LETTERS, 1997, 405 (01) :26-30
[4]   Expression patterns of class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol dehydrogenase in local retinoic acid synthesis [J].
Ang, HL ;
Deltour, L ;
ZgombicKnight, M ;
Wagner, MA ;
Duester, G .
ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH, 1996, 20 (06) :1050-1064
[5]   Retinoic acid synthesis in mouse embryos during gastrulation and craniofacial development linked to class IV alcohol dehydrogenase gene expression [J].
Ang, HL ;
Deltour, L ;
Hayamizu, TF ;
ZgombicKnight, M ;
Duester, G .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (16) :9526-9534
[6]  
BENSON S A, 1984, Biotechniques, V2, P126
[7]   PHYSIOLOGICAL SUBSTRATES FOR RAT ALCOHOL-DEHYDROGENASE CLASSES - ALDEHYDES OF LIPID-PEROXIDATION, OMEGA-HYDROXYFATTY ACIDS, AND RETINOIDS [J].
BOLEDA, MD ;
SAUBI, N ;
FARRES, J ;
PARES, X .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1993, 307 (01) :85-90
[8]   ROLE OF EXTRAHEPATIC ALCOHOL-DEHYDROGENASE IN RAT ETHANOL-METABOLISM [J].
BOLEDA, MD ;
JULIA, P ;
MORENO, A ;
PARES, X .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1989, 274 (01) :74-81
[9]  
Cleland W W, 1979, Methods Enzymol, V63, P103
[10]   FUNDAMENTAL MOLECULAR DIFFERENCES BETWEEN ALCOHOL-DEHYDROGENASE CLASSES [J].
DANIELSSON, O ;
ATRIAN, S ;
LUQUE, T ;
HJELMQVIST, L ;
GONZALEZDUARTE, R ;
JORNVALL, H .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (11) :4980-4984