Fructose 1,6-bisphosphatase from rabbit liver. [18O]Phosphate-water exchange as a probe of the catalytic mechanism

Sharp, Thomas R.; Benkovic, Stephen J.

doi:10.1021/bi00580a036

Cited by 16 publications

(8 citation statements)

References 39 publications

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“…The rate of binding of phosphate to the D-ADP complex can be calculated from the rate of loss of the fully labeled phosphate species at saturating ADP. The first-order disappearance of P1804 is described by k_2k+3/(k_2 + k+2) (Sharp & Benkovic, 1979) or P¡k.3 (Scheme I). Dividing the observed rate by the partition coefficient and by the dynein concentration yields a rate for phosphate binding of 8000 M'1 s'1.…”

Section: Resultsmentioning

confidence: 99%

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ADP release is rate limiting in steady state turnover by the dynein adenosinetriphosphatase

Holzbaur

Johnson

1989

Biochemistry

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The kinetics of the product release steps in the pathway of ATP hydrolysis by dynein were investigated by examining the rate and partition coefficient of phosphate-water 18O exchange under equilibrium and steady-state conditions. Dynein catalyzed both medium and intermediate phosphate-water oxygen exchange with a partition coefficient of 0.30. The dependence of the rate of loss of the fully labeled phosphate species on the concentration of ADP was hyperbolic, with an apparent Kd for the binding of ADP to dynein of 0.085 mM. The apparent second-order rate constant for phosphate binding to the dynein-ADP complex was 8000 M-1 s-1. The time course of medium phosphate-water oxygen exchange during net ATP hydrolysis was examined in the presence of an ATP regeneration system. The observed rate of loss of P18O4 was comparable to the rate observed at saturating ADP which implies that ADP release is rate limiting for dynein in the steady state. Product inhibition of the dynein ATPase was also examined. ADP inhibited the enzyme competitively with a Ki of 0.4 mM. Phosphate was a linear noncompetitive mixed-type inhibitor with a Ki of 11 mM. These data were fit to a model in which phosphate release is fast and is followed by rate-limiting release of ADP, allowing us to define each rate constant in the pathway. A discrepancy between the total free energy calculated compared to the known free energy of ATP hydrolysis suggests that there is an additional step in the pathway, perhaps involving a change in conformation of the enzyme-ADP state preceding ADP release.

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Section: Resultsmentioning

confidence: 99%

“…°C. The isotopic cluster at m/e 99 was monitored by a modification of the method ofSharp and Benkovic (1979).…”

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ADP release is rate limiting in steady state turnover by the dynein adenosinetriphosphatase

Holzbaur

Johnson

1989

Biochemistry

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“…98% of one ion (see Table II and below for discription of fragmentation pattern). The amount of P¡ -H2180 equilibration after 5 s was calculated from the data and Scheme II of Sharp & Benkovic (1979) where kon = 2.3 mM'1 s_1 and kex/k0ff = 1.4. This equilibration redistributes the major ion as shown under predicted values in Tables III and IV.…”

Section: Resultsmentioning

confidence: 99%

“…By monitoring the ion intensities in the molecular ion region of m/e 99, 101, 103, 105, and 107, which correspond to the (HO)4P+ fragment, one can calculate the percentages of unlabeled and higher labeled species. Sharp & Benkovic (1979) have described the protocol for monitoring the intensities of five m/e values. Synthesis.…”

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Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

Domanico

Rahil

Benkovic³

1985

Biochemistry

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The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.

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“…Pertinent to a more satisfactory resolution of this problem is the observation that Fru-P,ase from rabbit liver cqtalyzes the solvent exchange of l 8 O from initially highly enriched Pi (115). The exchange occurs with either Mn2+ or Mgz+ as the cofactor and requires the presence of fru-6-P.…”

Section: B L8o Exchangementioning

confidence: 99%

Mechanism of Action of Fructose 1,6‐Bisphosphatase

Benkovic

DeMaine

1982

Advances in Enzymology - And Related Areas of Molecular Biology

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Fructose 1,6-bisphosphatase from rabbit liver. [18O]Phosphate-water exchange as a probe of the catalytic mechanism

Cited by 16 publications

References 39 publications

ADP release is rate limiting in steady state turnover by the dynein adenosinetriphosphatase

ADP release is rate limiting in steady state turnover by the dynein adenosinetriphosphatase

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

Mechanism of Action of Fructose 1,6‐Bisphosphatase

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