The role of thiol groups in the structure and mechanism of action of arginine kinase.
1. A detailed study of the reaction of iodoacetamide with arginine kinase has been carried out. 2. The enzyme contains five reactive thiol groups per 37000g. of protein, all of which can be alkylated. 3. Below pH8.5 loss of activity is substantially independent of pH and can be correlated with the alkylation of a single pH-independent thiol. 4. One catalytic site per enzyme molecule is inferred. 5. The progress curves of the alkylation reaction are polyphasic and reveal a pH-and time-dependent sequential release of thiols which is dependent upon the alkylation of the first pH-independent thiol. This is supported by electrophoretic investigations. 6. Comparison of alkylation rate and rate of loss of activity suggests that two thiol groups are not essential for catalytic activity. Variability in enzyme preparations with respect to alkylation rate appears to be associated with these two groups. 7. A complex protection pattern is revealed by the effects of various substrate combinations on rates of alkylation and of loss of activity. It is inferred that two thiol groups participate in conformational changes and nucleotide interactions. 8. Comparison with creatine kinase suggests a fundamentally similar catalytic mechanism, although for arginine kinase certain additional restrictions are necessary because of the protection observed with nucleotide substrates.
D000227:Adenine Nucleotides; D000255:Adenosine Triphosphate; D000478:Alkylation; D000577:Amides; D001120:Arginine; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003402:Creatine Kinase; D004563:Electrochemistry; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007454:Iodides; D007461:Iodoacetates; D008274:Magnesium; D010770:Phosphotransferases / Q000032:analysis; D013696:Temperature
D000227:Adenine Nucleotides; D000577:Amides; D007454:Iodides; D007461:Iodoacetates; D000255:Adenosine Triphosphate; D001120:Arginine; D010770:Phosphotransferases; D003402:Creatine Kinase; D008274:Magnesium