Isozyme Multiplicity with Anomalous Dimer Patterns in a Class III Alcohol Dehydrogenase. Effects on the Activity and Quaternary Structure of Residue Exchanges at "Nonfunctional" Sites in a Native Prot
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- 作者：Olle Danielsson ; Jawed Shafqat ; Mats Estonius ; Mustafa El-Ahmad ; and Hans Jö ; rnvall
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：November 19, 1996
- 年：1996
- 卷：35
- 期：46
- 页码：14561 - 14568
- 全文大小：330K
- 年卷期：v.35,no.46(November 19, 1996)
- ISSN：1520-4995
文摘
The isozymes of class III alcoholdehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited threeunexpected properties of general interest.First, these dimeric isozymes, derived from two types of subunit(h and l, for high- and low-activityforms), were recovered from liver preparations in only the homodimericll and heterodimeric hlcombinations. Dissociation and reassociation of the isolatedhl form in vitro also resulted in loweryieldsof the hh than the ll homodimer, although classIII subunits are usually freely associable over widebordersof divergence (human and Drosophila). The hand l primary structures show that both chain typesarecharacteristic of class III enzymes, without large amino acidreplacements at positions of known subunitinteractions. Hence, the hh dimer partial restrictionindicates nontraditional alterations ath-subunitinterfaces. The structure provides a possible explanation, in theform of h-chain modifications that mayinfluence the anchoring of a loop at positions of two potentiallydeamidative 
-aspartyl shifts at distantAsn-Gly structures. Second, the ll and hlforms differ in enzymatic properties, having 5-folddifferentKm values for NAD+ at pH 8,different Km values forS-(hydroxymethyl)glutathione (10 versus 150
M),and different specific activities (4.5 versus 41 units/mg), withll resembling and hl deviating fromhumanand other class III alcohol dehydrogenases. However, functionalresidues lining substrate and coenzymepockets in the known conformations of homologous forms are largelyidentical in the two isozymes [onlyminor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, andIle/Val269 (numbering systemof the human class I enzyme)], again indicating effects from distantlypositioned h-chain replacements.Third, the two isozymes differ a surprising amount in amino acidsequence (18%, the same as the piscine/human difference), reflecting a remarkably old isozyme duplication or,more probably, discordantaccumulation of residue exchanges with greater speed of evolution forone of the subunits (h chain) thanis typical for the slowly evolving class III alcoholdehydrogenase.
-aspartyl shifts at distantAsn-Gly structures. Second, the ll and hlforms differ in enzymatic properties, having 5-folddifferentKm values for NAD+ at pH 8,different Km values forS-(hydroxymethyl)glutathione (10 versus 150M),and different specific activities (4.5 versus 41 units/mg), withll resembling and hl deviating fromhumanand other class III alcohol dehydrogenases. However, functionalresidues lining substrate and coenzymepockets in the known conformations of homologous forms are largelyidentical in the two isozymes [onlyminor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, andIle/Val269 (numbering systemof the human class I enzyme)], again indicating effects from distantlypositioned h-chain replacements.Third, the two isozymes differ a surprising amount in amino acidsequence (18%, the same as the piscine/human difference), reflecting a remarkably old isozyme duplication or,more probably, discordantaccumulation of residue exchanges with greater speed of evolution forone of the subunits (h chain) thanis typical for the slowly evolving class III alcoholdehydrogenase.