The use of arginine analogues for investigating the functional organization of the arginine-binding site in lobster muscle arginine kinase. Role of the 'essential' thiol group.
1. The nature of arginine binding to lobster arginine kinase and the extent of its possible involvement with the ;essential' thiol group of the enzyme has been investigated with some inhibitory analogues of arginine. 2. Most of the analogues inhibit competitively, although mixed inhibition may occur if the alpha-carboxy group or alpha-amino group is absent. 3. The K(i) values indicate that strength of binding depends on the length of the carbon chain (l-isoleucine>l-valine>l- alpha-aminobutyrate>l-alanine) and the integrity of the substituents on the alpha-carbon atom (l-arginine>agmatine and l-ornithine>putrescine). The guanidino group probably contributes little to substrate binding, but a positive charge near the delta-nitrogen atom appears to be important (l-ornithine>l -citrulline>l-alpha-aminobutyrate). A cyclic analogue, 2-carboxymethyl-3-oxo-2,3,5,6,7,8-hexahydro-1H-imidazo [1,2-a][1,3]diazepine-8-carboxylic acid, has a low K(i) value similar to that of an equivalent straight-chain form, suggesting that arginine probably binds in a folded configuration. 4. The aliphatic l-amino acids give enzyme difference spectra similar to that with l-arginine and the integrity of the alpha-carboxy and alpha-amino groups appears to be a minimal but not sufficient requirement for this, as l-ornithine gives an atypical difference spectrum. A difference spectrum is interpreted as indicating an enzyme conformational change. No difference spectrum was observed with methylguanidine. 5. The ability of aliphatic alpha-l-amino acids to protect against inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) is proportional to the number of atoms in the carbon chain and inversely proportional to K(i). Ornithine gives greater protection than citrulline; analogues lacking the alpha-amino groups also protect. Agmatine, lacking the alpha-carboxy group, did not protect. 6. It is concluded that it is unlikely that the ;essential' thiol group in the enzyme interacts with any part of the arginine molecule during catalysis except, possibly, the alpha-carboxyl group.
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism*; D001122:Arginine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D008121:Nephropidae / Q000201:enzymology; D010770:Phosphotransferases / Q000378:metabolism*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism
D013438:Sulfhydryl Compounds; D001120:Arginine; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D001122:Arginine Kinase