Analysis of positional isotope exchange in ATP by cleavage of the beta P-O gamma P bond. Demonstration of negligible positional isotope exchange by myosin.

A method for analysis of positional isotope exchange (PIX) during ATP in equilibrium with HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the beta P-O gamma P bond. This cleavage yields Pi derived from the gamma-phosphoryl of ATP that contains all four of the gamma oxygens. Both PIX between the beta,gamma-bridge and beta-nonbridge positions and washout of the gamma-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17O at the beta-nonbridge positions and 18O at the beta,gamma-bridge and gamma-nonbridge positions. Application of this method to ATP in equilibrium with HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of gamma-nonbridge oxygens in the virtual absence of PIX. At 25 degrees C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s-1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the beta-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 degrees C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. [Geeves, M. A., Webb, M. R., Midelfort, C. F., & Trentham, D. R. (1980) Biochemistry 19, 4748-4754] with subfragment 1.

[1]  M. Cohn.  18O and 17O effects on 31P NMR as probes of enzymatic reactions of phosphate compounds. , 1982, Annual review of biophysics and bioengineering.

[2]  P. Boyer,et al.  Refinements in oxygen-18 methodology for the study of phosphorylation mechanisms. , 1986, Methods in enzymology.

[3]  G. Lienhard,et al.  P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. , 1973, The Journal of biological chemistry.

[4]  P. Boyer,et al.  The equivalence of phosphate oxygens for exchange and the hydrolysis characteristics revealed by the distribution of [18O]Pi species formed by myosin and actomyosin ATPase. , 1980, The Journal of biological chemistry.

[5]  Clive R. Bagshaw,et al.  The reversibility of adenosine triphosphate cleavage by myosin. , 1973, The Biochemical journal.

[6]  D. Hackney,et al.  Steady state kinetics at high enzyme concentration. The myosin MgATPase. , 1985, The Journal of biological chemistry.

[7]  John H. Young,et al.  Pseudorotation mechanism of ATP hydrolysis in muscle contraction , 1974, Nature.

[8]  S. Lowey,et al.  [7] Preparation of myosin and its subfragments from rabbit skeletal muscle , 1982 .

[9]  P. Boyer,et al.  Oxygen-18 probes of enzymic reactions of phosphate compounds. , 1980, Methods in enzymology.

[10]  P. Boyer,et al.  Effect of actin concentration on the intermediate oxygen exchange of myosin; relation to the refractory state and the mechanism of exchange. , 1978, Biochemistry.

[11]  J. G. Moffatt,et al.  DISMUTATION REACTIONS OF NUCLEOSIDE POLYPHOSPHATES. II. SPECIFIC CHEMICAL SYNTHESES OF ALPHA-, BETA-, AND GAMMA-P32-NUCLEOSIDE 5'-TRIPHOSPHATES. , 1965, Journal of the American Chemical Society.

[12]  H. Khorana,et al.  Pyrophosphorylation of ribose 5-phosphate in the enzymatic synthesis of 5-phosphorylribose 1-pyrophosphate. , 1958, The Journal of biological chemistry.

[13]  D. Trentham,et al.  Mechanism of adenosine 5'-triphosphate cleavage by myosin: studies with oxygen-18-labeled adenosine 5'-triphosphate. , 1980, Biochemistry.

[14]  E. Eisenberg,et al.  The mechanism of the skeletal muscle myosin ATPase. II. Relationship between the fluorescence enhancement induced by ATP and the initial Pi burst. , 1979, The Journal of biological chemistry.

[15]  N. Oppenheimer,et al.  Enzyme-catalyzed positional isotope exchange by phosphorus-31 nuclear magnetic resonance spectroscopy using either oxygen-18- or oxygen-17-.beta.,.gamma.-bridge-labeled adenosine 5'-triphosphate , 1983 .

[16]  D. Hackney.  Theoretical analysis of distribution of [18O]Pi species during exchange with water. Application to exchanges catalyzed by yeast inorganic pyrophosphatase. , 1980, The Journal of biological chemistry.

[17]  D. Trentham,et al.  The stereochemical course of phosphoric residue transfer catalyzed by sarcoplasmic reticulum ATPase. , 1981, The Journal of biological chemistry.

[18]  R. Starr,et al.  A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. , 1973, Journal of molecular biology.

[19]  D. Hackney,et al.  Anomalous oxygen-18 exchange during ATP synthesis in oxidative phosphorylation. , 1986, Biochemistry.

[20]  R. Switzer,et al.  Phosphoribosylpyrophosphate synthetase (ribose-5-phosphate pyrophosphokinase) from Salmonella typhimurium. , 1978, Methods in enzymology.

[21]  I. A. Rose,et al.  Mechanism for oxygen exchange in the chloroplast photophosphorylation system. , 1977, The Journal of biological chemistry.

[22]  D. Trentham,et al.  The stereochemical course of phosphoric residue transfer during the myosin ATPase reaction. , 1980, The Journal of biological chemistry.

[23]  M. Webb.  A method for determining the positional isotope exchange in a nucleoside triphosphate: cyclization of nucleoside triphosphate by dicyclohexylcarbodiimide. , 1980, Biochemistry.

[24]  Clive R. Bagshaw,et al.  Oxygen exchange in the gamma-phosphoryl group of protein-bound ATP during Mg2+-dependent adenosine triphosphatase activity of myosin. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Boyer,et al.  Subunit interaction during catalysis. Alternating site cooperativity of mitochondrial adenosine triphosphatase. , 1979, The Journal of biological chemistry.

[26]  I. A. Rose.  Positional isotope exchange studies of enzyme mechanisms. , 1979, Advances in enzymology and related areas of molecular biology.

[27]  E. Taylor.  Transient phase of adenosine triphosphate hydrolysis by myosin, heavy meromyosin, and subfragment 1. , 1977, Biochemistry.

[28]  C. Grubmeyer,et al.  The stereochemical course of phosphoric residue transfer catalyzed by beef heart mitochondrial ATPase. , 1980, The Journal of biological chemistry.