Abstract
A nucleoside phosphotransferase has been purified to homogeneity from barley seedlings. Its Mr is about 50,000 and it consists of two subunits of equal size. A tightly bound metal ion required for activity can be replaced by Mg, Ba, or Co ions, but not by divalent Cu, Ca, Cd, or Hg. The enzyme is capable of catalyzing the transfer of phosphate from nucleoside monophosphates to the 5'-hydroxyl of any other nucleoside, but shows a decided preference for purine deoxynucleoside phosphate acceptors. A short lived phosphoryl enzyme intermediate has been trapped by rapid denaturation in presence of [32P]AMP. The steady state kinetics of this enzyme is found to be fully consistent with a branched ping pong pathway involving a compulsory phosphoryl enzyme intermediate from which phosphate may be transferred to nucleoside or to water. With appropriately chosen substrates, parallel line reciprocal plots were obtained, provided that both products of the branched pathway were taken into account in the rate measurements. Conversely, when only transphosphorylation was measured, converging reciprocal plots were obtained. Accordingly, assays were devised for the measurement of three velocity components: transphosphorylation, hydrolysis, and the sum of both reactions. Competitive inhibition of the transferase reaction by higher concentrations of either substrate is attributed to deadend interactions. Instead of being inhibited by the formation of a putative E-P.AMP complex, however, the phosphatase reaction is accelerated almost 2-fold.
Highlights
The steady state kinetics of this enzyme is found to be fullyconsistent with a branched ping pong pathway involving a compulsory phosphoryl enzyme intermediate from which phosphate may be transferredto nucleoside or towater
A recent stereochemical study reported from these laboratories showed that [’80]thiophosphoryl transfer catalyzed by the nucleoside phosphotransferase from barley proceeds with net retention of configuration, suggesting a double displacement mechanism involving the compulsory formation of a phosphoryl enzyme as anintermediate (3).We report the first direct chemical evidence for a covalently bonded [”‘PI
We have investigated the nucleoside phosphotransferase frombarley and found the kinetics to be consistent with a modified ping-pong kinetic pathway involving a hydrolytically labile phosphoryl enzyme
Summary
CHARACTERIZATION AND EVIDENCE FOR PING PONG KINETICS INVOLVING PHOSPHORYL ENZYME*. Douglas C. The steady state kinetics of this enzyme is found to be fullyconsistent with a branched ping pong pathway involving a compulsory phosphoryl enzyme intermediate from which phosphate may be transferredto nucleoside or towater. Steady state rateanalyses of the nucleoside phosphotransferases from carrot (1) and fern (2) yielded converging initial rate patterns in double reciprocal plots. Such patterns are usually associated with sequential kinetic pathways, as distinguished from double displacement, i.e. ping-pong, pathways. These enzymes exhibit inherent nucleotidase activities, which complicates the interpretation of conventional plots of initial rate data; these complicationswere not taken into account in the earlier work. We have detected several enzyme-substrate interactions in addition to those represented by Equations 1 to 3
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