1. The initial formation of arginine phosphate by arginine kinase was studied in the time range 2.8 -50 ms by the quenched-flow method.2. A transient burst phase of product formation was obtained, the amplitude of which was temperature-dependent. At 35 "C it was 0.64 mol arginine phosphate/mol arginine kinase and at 12 "C, 0.25 mol/mol.3. These results show that for the reaction pathway of arginine kinase the rate-limiting step follows the formation of arginine phosphate on the enzyme. This is in contrast to the creatine kinase reaction where 4. The rate-limiting step on the arginine kinase reaction pathway is only slightly affected by temperature: the change in k,,, with temperature is due to a change of an equilibrium constant pertaining to at least two previous steps.There is at present little information on the reaction mechanisms of the phosphagen kinases, indeed on the phosphoryl transferases in general (see, for example, [l, 21). The elucidation of the mechanisms of action of the phosphagen kinases is of some importance as they occupy a central position in muscle contraction and metabolism. The best-studied enzymes of this group are creatine kinase and arginine kinase; these enzymes catalyze the synthesis of creatine phosphate and arginine phosphate, respectively :Creatine + ATP Creatine phosphate + ADP Arginine + ATP * Arginine phosphate + ADP.Creatine kinase is the phosphagen kinase of vertebrate muscle ; arginine kinase is found in invertebrates such as the lobster (Homarus vulgaris) [3,4].Both enzymes have a rapid-equilibrium random type of mechanism [3,4]. This indicates that the ratelimiting steps in their reaction pathways occur after the formation of the ternary complex enzyme . ATP . guanidine. These studies are in agreement with the relaxation work of Hammes and Hurst [5] who found that, with creatine kinase, the steps characterizing the binding of the substrates are considerably faster than the turnover of the overall reaction. More recent work, however, suggests that the mechanisms of action of the two enzymes may be different. Thus, whereas the nuclear magnetic resonance studies of Rao et al.[6] show that the chemical step in the arginine kinase reaction pathway is fast and not rate-limiting, the quenched-flow studies of Engelborghs et al. [7] show that the same step in the creatine kinase pathway is slow and rate-limiting. These differences may indicate basic differences in the mechanisms of creatine and arginine kinase; on the other hand, they may merely indicate differences in the relative importance of the rate constants associated with rate-limiting steps.Information on the mechanisms of the phosphagen kinases is hard to come by. The formation and interconversion of the intermediates on their reaction pathways are not, in general, accompanied by distinct optical signals. This precludes the use of methods such as the stopped-flow technique. Furthermore, with the exception of creatine and arginine kinases, these enzymes are difficult to obtain in the quantities required by most rapid-rea...

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The early steps of the Mg(2+)-ATPase activity of relaxed rabbit psoas myofibrils were studied in a buffer of near-physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase, and the cleavage and release of product steps by the Pi burst method. The data obtained were interpreted by [formula: see text] where M represents the myosin heads with or without actin interaction. This work is a continuation of our study on Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. Here the constants obtained with relaxed myofibrils were compared with those with activated myofibrils and myosin subfragment 1 (S1). We find that whereas Ca2+ increases 80X the release of products (k4), it has little effect upon the kinetics of the initial binding and cleavage steps. As with activated myofibrils and S1, the second-order binding constant for ATP (k2/K1) was about 1 microM-1 s-1 and the ATP was bound very tightly. With activated myofibrils, it was difficult to obtain an estimate for the koff for ATP(k-2) but it is much less than kcat. Here with relaxed myofibrils we estimate k-2 less than 8 x 10(-4) s-1, which is considerably smaller than kcat (0.019 s-1) and also previous estimates for this constant. The overall Kd for ATP to relaxed myofibrils is less than 8 x 10(-10) M. With S1 this Kd is about 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)

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