Document Detail

Modeling of a mutation responsible for human 3-hydroxy-3-methylglutaryl-CoA lyase deficiency implicates histidine 233 as an active site residue.
MedLine Citation:
PMID:  8798725     Owner:  NLM     Status:  MEDLINE    
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude. Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.
J R Roberts; G A Mitchell; H M Miziorko
Related Documents :
11136555 - Changes in the carboxyl terminus of the beta subunit of human propionyl-coa carboxylase...
11099795 - Kinetic behavior of the pancreatic lipase-colipase-lipid system.
1732385 - Structural basis for the barrier abnormality following inhibition of hmg coa reductase ...
17442255 - Assessing the roles of essential functional groups in the mechanism of homoserine succi...
10101215 - The unique properties of dipeptidyl-peptidase iv (dpp iv / cd26) and the therapeutic po...
21375505 - Substrate diffusion and oxidation in gmc oxidoreductases: an experimental and computati...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  271     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1996 Oct 
Date Detail:
Created Date:  1996-11-25     Completed Date:  1996-11-25     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  24604-9     Citation Subset:  IM    
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Acyl Coenzyme A / metabolism
Binding Sites
Diethyl Pyrocarbonate / pharmacology
Enzyme Inhibitors / pharmacology
Histidine / metabolism*
Metabolism, Inborn Errors / enzymology,  genetics*
Oxo-Acid-Lyases / antagonists & inhibitors,  genetics*,  metabolism
Point Mutation*
Grant Support
Reg. No./Substance:
0/Acyl Coenzyme A; 0/Enzyme Inhibitors; 1553-55-5/3-hydroxy-3-methylglutaryl-coenzyme A; 1609-47-8/Diethyl Pyrocarbonate; 71-00-1/Histidine; EC 4.1.3.-/Oxo-Acid-Lyases; EC A lyase

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

Previous Document:  Rescue of His-42 --> Ala horseradish peroxidase by a Phe-41 --> His mutation. Engineering of a surro...
Next Document:  Substitution of glutamic 779 with alanine in the Na,K-ATPase alpha subunit removes voltage dependenc...