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Structural Basis for Substrate Specificity Differences of Horse Liver Alcohol Dehydrogenase Isozymes

Zwart, Peter; Adolph, Hans-Werner; Meijers, Rob; Zwart, Peter; Hubatsch, Ina; Kiefer, Martin; Meijers, Rob; Lamzin, Victor; Cedergren, Eila LU and Hubatsch, Ina, et al. (2000) In Biochemistry 39(42). p.12885-12897
Abstract
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, 5β-androstane-3β,17β-ol, 5β-androstane-17β-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 3α,7α,12α-trihydroxy-5β-cholan-24-acid (cholic acid) and NAD+, but microspectrophotometric analysis of single crystals... (More)
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, 5β-androstane-3β,17β-ol, 5β-androstane-17β-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 3α,7α,12α-trihydroxy-5β-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 P21 with cell dimensions a = 55.0 Å, b = 73.2 Å, c = 92.5 Å, and β = 102.5. A 98% complete data set to 1.54-Å 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 Cα carbon positions (about 5 Å) 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. (Less)
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Biochemistry
volume
39
issue
42
pages
12885 - 12897
publisher
The American Chemical Society
external identifiers
  • scopus:0034711039
ISSN
0006-2960
DOI
10.1021/bi001376s
language
English
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yes
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c1ee65f0-f41d-4a2f-bdb7-8670a222874f (old id 125067)
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2007-06-26 09:45:01
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2017-11-12 03:23:20
@article{c1ee65f0-f41d-4a2f-bdb7-8670a222874f,
  abstract     = {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, 5β-androstane-3β,17β-ol, 5β-androstane-17β-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 3α,7α,12α-trihydroxy-5β-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 P21 with cell dimensions a = 55.0 Å, b = 73.2 Å, c = 92.5 Å, and β = 102.5. A 98% complete data set to 1.54-Å 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 Cα carbon positions (about 5 Å) 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.},
  author       = {Zwart, Peter and Adolph, Hans-Werner and Meijers, Rob and Zwart, Peter and Hubatsch, Ina and Kiefer, Martin and Meijers, Rob and Lamzin, Victor and Cedergren, Eila and Hubatsch, Ina and Kiefer, Martin and Lamzin, Victor},
  issn         = {0006-2960},
  language     = {eng},
  number       = {42},
  pages        = {12885--12897},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Structural Basis for Substrate Specificity Differences of Horse Liver Alcohol Dehydrogenase Isozymes},
  url          = {http://dx.doi.org/10.1021/bi001376s},
  volume       = {39},
  year         = {2000},
}