Transient-Phase Studies on the Arginine Kinase Reaction

Abstract

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...

An Attachment for Nondestructive, Fast Quenching of Samples in Rapid-Mixing Experiments

“…The quantitative description of the time courses of temperature or of concentration for the simplest possible case is given by Eqs. [1] through [3]. Here we consider that mixing is instantaneous.…”

“…Equations [2] and [3] define conditions for optimizing quenching and washing in the QWC by cooling and dilution. They indicate the relationship between the values of F R /F and of the chamber volume, V, that will give minimum increases in the value of X and the quickest return of X to X Q .…”

“…Many biological processes such as enzymatic reactions in pre-steady state and rapid membrane transport take place in time spans of the order of milliseconds (1)(2)(3)(4). Rapid-mixing devices make it possible to initiate and terminate a process in lengths of time that are short compared to the time constant of the process to be measured (5,6).…”

“…Stopped flow can be applied when it is possible to obtain a signal from the reaction mixture to monitor a process. Otherwise, quenched flow, which is the arrest of a process by means of a sudden change in the chemical and/or physical properties of the medium, must be applied (3,6).…”

Cryoenzymological Studies on Myosin Subfragment 1

“…The quenched reaction mixtures were diluted to 2.5 ml by the addition of 1.25 ml 0.2 M HzS04, 0.24 M magnesium perchlorate and the protein removed by centrifugation. 2 ml of the supernatant was removed and the inorganic phosphate determined as described previously [14].…”