Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes.
A structure determination in combination with a kinetic study of the steroid converting isozyme of horse liver alcohol dehydrogenase, SS-ADH, is presented. Kinetic parameters for the substrates, 5beta-androstane-3beta,17beta-ol, 5beta-androstane-17beta-ol-3-one, ethanol, and various secondary alcohols and the corresponding ketones are compared for the SS- and EE-isozymes which differ by nine amino acid substitutions and one deletion. Differences in substrate specificity and stereoselectivity are explained on the basis of individual kinetic rate constants for the underlying ordered bi-bi mechanism. SS-ADH was crystallized in complex with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan -24-acid (cholic acid) and NAD(+), but microspectrophotometric analysis of single crystals proved it to be a mixed complex containing 60-70% NAD(+) and 30-40% NADH. The crystals belong to the space group P2(1) with cell dimensions a = 55.0 A, b = 73.2 A, c = 92.5 A, and beta = 102.5 degrees. A 98% complete data set to 1.54-A resolution was collected at 100 K using synchrotron radiation. The structure was solved by the molecular replacement method utilizing EE-ADH as the search model. The major structural difference between the isozymes is a widening of the substrate channel. The largest shifts in C(alpha) carbon positions (about 5 A) are observed in the loop region, in which a deletion of Asp115 is found in the SS isozyme. SS-ADH easily accommodates cholic acid, whereas steroid substrates of similar bulkiness would not fit into the EE-ADH substrate site. In the ternary complex with NAD(+)/NADH, we find that the carboxyl group of cholic acid ligates to the active site zinc ion, which probably contributes to the strong binding in the ternary NAD(+) complex.
Adolph
H W
HW
Fachrichtung 8.8 Biochemie, Universität des Saarlandes, D-66041 Saarbrücken, Federal Republic of Germany. hwadolph@rz.uni-sb.de
Zwart
P
P
Meijers
R
R
Hubatsch
I
I
Kiefer
M
M
Lamzin
V
V
Cedergren-Zeppezauer
E
E
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D019826:Cholic Acid / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004583:Electrons; D006736:Horses; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D015591:Microspectrophotometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007527:Isoenzymes; D009243:NAD; D000426:Alcohol Dehydrogenase; D019826:Cholic Acid