Myokinase reaction

Nevertheless, there is enough creatine phosphate to sustain only a few seconds of intensive work. Another system available for ATP generation is the myokinase reaction ... 

The purine nucleotide cycle also is involved in muscle energy production. During intense stimulation, or when O2 supply is limited, the high-energy bond of ADP is used to synthesize ATP via the myokinase reaction (Figure 21-12). The resulting AMP can dephosphorylate to adenosine, which diffuses out of the cell. Conversion of AMP to IMP via adenylate deaminase and then to adenylosuccinate helps sustain the myokinase reaction, especially in FG fibers, by reducing accumulation of AMP. It may also reduce the loss of adenosine from the cell, since nucleosides permeate cell membranes while nucleotides do not. 

The first equation is a reflection of the energy needed to maintain the brain function (ATPase) when the production of ATP by oxidative phosphorylation is compromised the second (creatine phosphokinase) and third (myokinase) reactions are secondary pathways attempting to maintain the ATP levels. In addition, ATP can be produced by anaerobic glycolysis and this explains the relative depletion of the glucose-related metabolites and a tenfold increase in lactate with a resulting acidosis (data not shown.). 

Anaerobic production of ATP by substrate-level phosphorylation, from phosphocreatine and by the adenyiate kinase (myokinase) reaction... 

In this way NAD is regenerated and the muscle cells can continue to produce small amounts of ATP glycolytically. ATP may also be derived from phosphocreatine and from the myokinase reaction. 

Living systems have still another mechanism for making ADP available as a phosphate acceptor and that is the myokinase reaction previously mentioned whereby 1 mole of ATP may react with 1 mole of AMP to yield 2 moles of ADP. Such a device then serves the purpose of regenerating ADP from ATP as long as a source of AMP is available. This enzyme will serve to keep the proper adenine nucleotides in desirable concentration as the needs of the cell dictate. 

ADP was also found to be a phosphate donor in the flavokinase reaction above, but the maximal velocity of formation of riboflavin 5 -phosphate was only about one-half that obtained with ATP. The Ks for ATP and ADP are, respectively, 1.7 X 10 M and 1.6 X 10 M (124). Englard (123) observed that purified flavokinase from brewer s yeast still contained myokinase, and attributed the activity of ADP to this contamination. He reported that in the presence of Zn++, when the myokinase contaminant is virtually inactive, flavokinase activity proceeded with ATP but not with ADP. However, there still appears to be some doubt whether ADP can be completely excluded as a phosphate donor in the flavokinase reaction on these grounds (124). Inosine triphosphate is inactive and adenoitine 6 -phosphate has been reported to be a competitive inhibitor of the reaction (121, 122). Ei ard (123) believes that the inhibition by adenosine 5 -phosphate is probably due to the competition of myokinase reaction with flavokinase for the available ATP when both reactions are activated by Mg++ adenosine 5 -phosphate did not inhibit flavokinase in the presence of Zn++ when myokinase was inactive. 

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

This enzyme [EC 2.7.4.3], also known as myokinase, catalyzes the reversible reaction of MgATP with AMP to produce MgADP and ADP. Inorganic triphosphate can also act as substrate with this enzyme. See Energy Charge Metal Ions in Nucleotide-Dependent Reactions... 

ADP as a substrate in enzyme reactions, ADENYLATE KINASE (or MYOKINASE) ATP SYNTHASE CREATINE KINASE NUCLEOSIDE DIPHOSPHATE KINASE PHOSPHOGLYCERATE KINASE PYRUVATE KINASE RIBONUCLEOTIDE REDUCTASE SULFATE ADENYLYLTRANSFERASE (ADP) [ADP]/[ATP] ratio,... 

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

Adenylate kinase (myokinase) catalyzes the following reversible reaction. 

With the presence of an AMP kinase in the complex established, it is necessary to determine whether an ATP pyrophosphohydrolase activity is present in the complex to catalyze reaction (2). For these experiments, a reaction mixture is prepared with unlabeled ATP only and the formation of AMP and ADP is followed. Since the myokinase is present, as well as any AMP formed from the pyrophosphohydrolase, the remaining ATP will be used by the myokinase, and ADP will be formed. Of course, this ADP might be formed directly by an ATPase according to reaction (1), and the AMP... 

Additional experiments were undertaken in which an inhibitor of myoki-nase activity was added to a similar reaction mixture. Incubations were again carried out, and samples were removed and analyzed after 20 minutes. Figure 10.5 shows profiles (three background chromatograms) in which the myokinase activity was progressively inhibited As inhibition of the myokinase increased, the amount of ADP recovered declined, and the amount of AMP increased proportionately. The area of each of the peaks (ADP and AMP) was determined, and these data (inset) illustrate the proportionality between the decline in ADP and the increase in AMP. Clearly, these results rule out the pathway for the formation of AMP from ADP, but they are consistent with the formation of ADP as a result of the combined actions of reactions (2) and (3). In addition, these data suggest that an ATPase activity is present and would account for the formation of the unlabeled ADP observed in the original experiment. 

The following method has been described for the assay of cyclic AMP [144, 145]. Cyclic AMP is converted to 5 -AMP with the aid of phosphodiesterase and then to ATP with myokinase (EC 2.V.4.3 ATP AMP phosphotransferase adenylate kinase) and pyruvate kinase (EC 2.7.1.40 ATP pyruvate phosphotransferase). ATP is measured by determining the orthophosphate which accumulates during incubation of ATP with a cycling system containing myosin, pyruvate kinase, and phosphoenol pyruvate. Alternately, the ATP is determined by its luminescent reaction with firefly luciferin and luciferase [145-147]. With a sensitivity to about 1 pmol/tube of cyclic AMP, this assay is almost as sensitive as the phosphorylase method, but with a linearity over three orders of magnitude, it is linear over a much wider range than the phosphorylase method. 

When ATP has been hydrolysed to provide energy for muscle contraction, ADP accumulates. Remember that ADP stiU has a source of imtapped energy in the a-phosphoanhydride bond (Fig. 10.1). With ingenious hiochemical resourcefulness, this energy is salvaged when two molecules of ADP form ATP under anaerobic conditions using the adenylate kinase reaction (previously known as myokinase) (Fig. 10.5). 

Myokinase (adenylate kinase), specific for adenine nucleotides, was the first of the nucleotide monophosphokinases to be discovered 110, 111), The enzyme catalyzes the following reaction ... 

The free energy of ADP cleavage can also be used. Muscle and other tissues contain the enzyme myokinase, vhich catalyzes the following reaction ... 

The regeneration system for CMP-NeuAc is more complicated than that for NDP-sugars (Scheme 7) [24]. An additional phosphorylation step must be incorporated, because CMP, a nucleoside monophosphate, is released after reaction with the sialyltransferase. For recycling purposes, nucleoside monophosphate kinase (NMK EC 2.7.4.4) or myokinase (MK EC 2.7.4.3) is added for the conversion of CMP to CDP. In this reaction, the phosphoryl donor is ATP. Subsequently, both CDP and ADP must be re-phosphorylated to CTP and ATP, respectively. Thus, for regeneration of CMP-NeuAc, an additional kinase and two equivalents of PEP are required. The condensation of NeuAc with CTP is catalyzed by CMP-NeuAc synthetase (EC 2.7.7.43). This system was used for the large-scale synthesis of 6 -sialyl-LacNAc(6 -SLN) from LacNAc catalyzed by a2,6-SiaT (EC 2.7.7.43) in 97% yield. 

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