The Essential Thiol Group of Arginine Kinase from Homarus vulgaris Muscle Studied by Difference Spectrophotometry and N‐Ethyl‐[l‐¹⁴C]Maleimide Labelling

Abstract

Interaction of N-ethylmaleimide with the essential cysteinyl residue of ATP : L-arginine phosphotransferase from Homarus vulgaris muscle has been studied by means of difference spectrophotometry and labelling with radioactive N-ethyl-[l-14C]maleimide.Cysteine and glutathione were used as model compounds reacting with the inhibitor. The difference spectrum at 238 nm observed with the L-arginine-arginine kinase complex might be ascribed to the modification of ionized -SH groups. The absorption change observed near 287nm, produced by tyrosyl perturbation, is the same when the enzyme reacts with the inhibitor or with L-arginine.Labelling data give evidence of a significant protection of the essential thiol group by L-arginine against N-ethyl-[ 1 -14C]maleimide. This cysteinyl residue is included in the tryptic peptide T,A the amino acid sequence of which, recently determined, is very similar to that of the homologous peptides of muscle and brain rabbit creatine kinases and of earth-worm muscle lombricine kinase .In previous papers [1-3] it was reported that cysteinyl residues were involved in the enzymic activity of arginine phosphotransferase. Furthermore Kassab et al. 141 have shown that the activity was related to the occurrence of one essential -SH group per 40000 g protein. It was assumed that the thiol group was hydrogen bound to a histidyl residue in the active site [3].The present paper is a part of difference spectrophotometry investigations by means of which the precise functions of essential amino acid residues in the catalytic process of the arginine kinase reaction are studied.The binding of L-arginine and nucleotide substrates to the enzyme produced characteristic difference spectra [5]. Evidence was given that the binding of L-arginine to arginine kinase occurred on or near ,COOH the essential -SH group through the -CH terminal of the substrate [5] since it protecting effect was afforded by L-arginine and various L-amino acids against sulfhydryl reagent inhibition and since alkylation of arginine kinase with iodoacetamide provided a difference spectrum similar to that produced with L-arginine and L-amino acids.