Inhibition and covalent modification of rape seed (Brassica napus) enoyl ACP reductase by phenylglyoxal.
The NADH-dependent enoyl-ACP reductase from oil seed rape (Brassica napus) was inactivated by treatment with phenylglyoxal, a reagent which specifically modifies arginine residues. The inhibition at various phenylglyoxal concentrations shows pseudo-first-order kinetics, with an apparent second-order rate constant of 14.2 M-1.min-1 for inactivation. The protective ability of several substrates and substrate analogues was investigated in order to ascertain if the inhibition was directed towards the active site of the enzyme. NADH and NAD+ did not protect but acyl carrier protein (ACP) and reduced coenzyme A, along with various derivatives, did protect. 9 microM ACP gave 35% protection from inactivation and 10 mM reduced coenzyme A gave 98% protection. The effectiveness of various subfragments of coenzyme A in protecting against inhibition indicates that the phosphate group is essential for preventing the binding of phenylglyoxal. The idea that phenylglyoxal is inhibiting by binding at the active site is further supported by the observation that the incorporation of 14C-labelled phenylglyoxal is directly related to the loss of activity. Extrapolation of the amount of label incorporated to give total inhibition shows that 4 mol of phenylglyoxal would be incorporated per mol of enzyme. This corresponds to the modification of two arginine side-chains with equal reactiveness towards the reagent. These results are consistent with there being two arginine residues either at the active site of the enzyme or in an environment which is protected from phenylglyoxal by a conformational change induced by coenzyme A binding.
Cottingham
I R
IR
Protein Chemistry Section, Unilever Research, Sharnbrook, U.K.
Austin
A J
AJ
Slabas
A R
AR
D000447:Aldehydes / Q000494:pharmacology*; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001937:Brassica / Q000201:enzymology*; D003545:Cysteine / Q000378:metabolism; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000447:Aldehydes; D001120:Arginine; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D003545:Cysteine; D010658:Phenylglyoxal