Studies on medium-chain fatty acyl-coenzyme A synthetase. Purification and properties

Abstract

1. Medium-chain fatty acyl-CoA synthetase (EC 6.2.1.2) was isolated by the method of Mahler, Wakil & Bock (1953) and the enzyme activity determined by the disappearance of CoA in the presence either of butyrate and ATP or of butyryl-AMP, as well as by ATP formation from butyryl-AMP and PP(i). 2. Preincubation of the enzyme with CoA and ATP alone or together, followed by the removal of these substrates by gel filtration, caused a marked inhibition of ATP formation, contrary to results previously obtained with palmitoyl-CoA synthetase. 3. The effect of ATP on butyryl-AMP-dependent CoA disappearance was inconsistent. Low concentrations of ATP (0.1-0.5mm) always caused inhibition, whereas higher concentrations (5-10mm) activated in some enzyme preparations and inhibited in others. 4. This inconsistency was shown to be due to the presence of two enzyme fractions. Both fractions had similar activities when assayed by the butyryl-AMP- or butyrate-plus-ATP-dependent CoA disappearance. However, fraction I was activated by ATP as measured by butyryl-AMP-dependent CoA disappearance whereas fraction II was inhibited by it. Fraction I also catalysed ATP formation from butyryl-AMP and PP(i) whereas fraction II was lacking in such activity. 5. The relationship of these observations with respect to other known mechanisms of fatty acid-activating systems is discussed.

Structural Snapshots for the Conformation-dependent Catalysis by Human Medium-chain Acyl-coenzyme A Synthetase ACSM2A

“…Our data represent the first structure determination for a human adenylate-forming enzyme, and provide the structural basis for its substrate preference and the molecular switch for the domain rearrangement that is central to the proposed catalytic mechanism. [25][26][27] …”

Catabolism of methylperhydroindanedione propionate by Rhodococcus equi: evidence of a MEPHIP-reductase activity

“…The measurement of this activity by determination of CoA levels using 5,5'-dithio-bis(2-nitrobenzyl) (DTNB) (Bar-Tana et al 1968) is not sufficiently sensitive…”

Studies of medium-chain fatty acyl-coenzyme A synthetase. Enzyme fraction II: mechanism of reaction and specific properties

“…1. The mechanism ofreaction of fatty acyl-CoA synthesis catalysed by fatty acyl-CoA synthetase from ox liver (fraction II; Bar-Tana, Rose & Shapiro, 1968) was investigated by a kinetic study of CoA disappearance dependent on butyrate plus ATP or butyryl-AMP (overall and partial reaction b respectively). 2.…”

“…Fractionation of an extract from ox liver particle acetone-dried powder results in two separate enzyme fractions I and II (Bar-Tana, Rose & Shapiro, 1968) capable of catalysing the synthesis of acyl-CoA from medium-chain fatty acids and CoA in the presence of ATP, according to the overall stoicheiometry determined by Mahler, Wakil & Bock (1953): R*CO2H+ATP+CoASH = R*CO *SCoA+PPi+AMP Studies on the mode of action of fraction I were described in the preceding paper (Bar-Tana & Rose, 1968). The overall reaction was found to proceed according to a mechanism similar to that proposed by Berg (1956) for yeast acetyl-CoA synthetase (Bi Uni Uni Bi Ping Pong according to Cleland, 1963a,b,c).…”

“…Such information, together with data relevant to the order of entry of the substrates and exit of the products of the reaction, would serve to clarify the relationship between the allosteric site and the active site of the enzyme. preceding papers (Bar-Tana et al 1968;Bar-Tana & Rose, 1968 and analyses of kinetic data were as described in the preceding paper (Bar-Tana & Rose, 1968).…”