Recombinant Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Purification, Characterization, and the Mechanism of Interactions with Functionally Diverse C8-CoA Molecules

Biochemistry ◽  
1995 ◽  
Vol 34 (45) ◽  
pp. 14942-14953 ◽  
Author(s):  
Kevin L. Peterson ◽  
Eduard E. Sergienko ◽  
Yihe Wu ◽  
N. Ravi Kumar ◽  
Arnold W. Strauss ◽  
...  
Keyword(s):  
Human Liver ◽  
Medium Chain ◽  
Chain Acyl ◽  
Functionally Diverse

scholarly journals Functional role of a distal (3′-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction

1997 ◽  
Vol 325 (3) ◽  
pp. 751-760 ◽  
Author(s):  
Kevin L. PETERSON ◽  
D. K. SRIVASTAVA
Keyword(s):  
Human Liver ◽  
Functional Role ◽  
Turnover Rate ◽  
Phosphate Group ◽  
Crystallographic Structure ◽  
Transient Kinetics ◽  
Enzyme Active Site ◽  
Medium Chain ◽  
Catalysed Reaction ◽  
Chain Acyl

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)–octenoyl-CoA complex reveals that the 3′-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 Å (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3′-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3′-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3′-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5–10-fold on deletion of the 3′-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation ‘off-rate’ (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3′-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3′-phosphate group of acyl-CoA substrates might seem ‘useless’ on the basis of the structural data, it has an essential functional role during HMCAD catalysis.

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