The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5°C, a transient pre-steady-state 'burst' of p-nitrophenol was formed with a rate constant of 48 s-1 The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. V(max) was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with k(cat) = 4.47 x 1013 s-1 at pH 5.0, 37°C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant K8 = 6.0 mM, the apparent K(m) = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37°C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1991|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology