Document Detail


Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+.
MedLine Citation:
PMID:  7988726     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp-39 is replaced with asparagine, has a 60-fold increase in affinity for NADP+ and a 1.5-fold increase in kcat compared to wild-type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263-267] and proposed that this part of ADH is close to the 2'-phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala-46 with an argine residue, and A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three-dimensional structure of the NAD(+)-binding region of ADH. The A46R mutant has a 2.5-fold lower Km(app)NADP+ and a 3-fold higher kcat with NADP+ compared to wild-type ADH; binding of NAD+ to the mutant was unchanged and kcat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of kcat/Km of NAD+ to NADP+ is 85, over ten-fold lower than that for wild-type ADH. Our model of the 3D structure of the NAD(+)-binding region of ADH shows that Ala-46 is over 10 A from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp-39 and the 2'-phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30-fold lower Km(app)NADP+ and 8-fold higher kcat with NADP+ as a cofactor compared to wild-type ADH; binding of NAD+ to this double mutant was lowered by 5-fold and kcat was increased by 1.5-fold. As a result, kcat/Km for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; kcat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.
Authors:
Z Chen; I Tsigelny; W R Lee; M E Baker; S H Chang
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  FEBS letters     Volume:  356     ISSN:  0014-5793     ISO Abbreviation:  FEBS Lett.     Publication Date:  1994 Dec 
Date Detail:
Created Date:  1995-01-11     Completed Date:  1995-01-11     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0155157     Medline TA:  FEBS Lett     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  81-5     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
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MeSH Terms
Descriptor/Qualifier:
Alanine / metabolism
Alcohol Dehydrogenase / chemistry,  genetics,  metabolism*
Animals
Arginine / metabolism
Codon
Drosophila / enzymology*
Electrochemistry
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
NADP / metabolism*
Protein Structure, Tertiary
Recombinant Proteins
Substrate Specificity
Grant Support
ID/Acronym/Agency:
ES03347/ES/NIEHS NIH HHS
Chemical
Reg. No./Substance:
0/Codon; 0/Recombinant Proteins; 53-59-8/NADP; 56-41-7/Alanine; 74-79-3/Arginine; EC 1.1.1.1/Alcohol Dehydrogenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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