Phosphoryl transfer pyruvate kinase

The second ATP-synthesizing reaction of glycolysis is catalyzed by pyruvate kinase, which brings the pathway at last to its pyruvate branch point. Pyruvate kinase mediates the transfer of a phosphoryl group from phosphoenolpyru-vate to ADP to make ATP and pyruvate (Figure 19.27). The reaction requires... 

Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-CoA, and alanine. (Note that alanine is the a-amino acid counterpart of the a-keto acid, pyruvate.) Furthermore, liver pyruvate kinase is regulated by covalent modification. Flormones such as glucagon activate a cAMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme. The phos-phorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher for PEP, so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the pathway (to be described in Chapter 23), instead... 

FIGURE 19.29 A mechanism for the pyruvate kinase reaction, based on NMR and EPR studies by Albert Mildvan and colleagues. Phosphoryl transfer from phosphoenolpyrnvate (PEP) to ADP occurs in four steps (a) a water on the Mg ion coordinated to ADP is replaced by the phosphoryl group of PEP (b) Mg dissociates from the -P of ADP (c) the phosphoryl group is transferred and (d) the enolate of pyruvate is protonated. (Adapted from Mildvan, A., 1979. Advances in Eiizymology 49 103-126.)... 

Transfer of the phosphoryl group to ADP in step 10 then generates ATP and gives enolpyruvate, which undergoes tautomerization to pyruvate. The reaction is catalyzed by pyruvate kinase and requires that a molecule of fructose 1,6-bis-phosphate also be present, as well as 2 equivalents of Mg2+. One Mg2+ ion coordinates to ADP, and the other increases the acidity of a water molecule necessary for protonation of the enolate ion. 

Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K+ and either Mg2+ or Mn2+ ... 

Kinases Direct transfer of terminal phosphoryl group of ATP to substrate Creatine kinase Adenylate kinase Hexokinasc Phosphoglycerate kinase Pyruvate kinase Protein kinase Myokinase Phosphofructokinase Type 1 (M-S-E) Type 1 Type 1 Type 1 Type 2 (S-M-E)... 

The A isomer of /3,-y-bidentate CrATP is the only form of this complex which activates the phosphoryl-transfer reaction of pyruvate kinase.281... 

Available evidence (14,15) favors the pathway for pyruvate kinase by way of phosphorylation of pyruvate enol. Furthermore, J. Knowles and his coworkers (16,17), using chiral thiophosphates and chiral (160,170,180) phosphate have shown that pyruvate kinase transfers phosphate from phosphoenolpyruvate to ADP with stereochemical inversion at phosphorus. Since monomeric metaphosphate is presumably planar, a chemical reaction by way of that ion should proceed with racemization. In the active site of an enzyme, however, all components might be held so rigidly that racemization need not occur. Furthermore, no information is yet available on the detailed mechanism of reactions catalyzed by cytidine synthetase our own experiments, designed to distinguish among the mechanisms here discussed, are as yet incomplete. 

I n this way we have shown that phosphoryl transfer catalysed by Bacillus stearothermophilus and rabbit skeletal muscle phosphofructokinase (6), and rabbit skeletal muscle pyruvate kinase occurs with inversion of configuration at phosphorus (7). The simplest interpretation of these stereochemical results is that phosphoryl transfer occurs by an in-line mechanism in the enzyme substrate ternary complexes. Stereochemical analysis is thus proving to be of considerable importance for delineating the mechanism adopted by phosphokinases. ... 

In the last reaction, pyruvate kinase catalyzes the physiologically irreversible transfer of the phosphoryl group from PEP to ADP to form ATP and pyruvate. 

The stereochemical consequence of [lsO]thiophosphoryl and [l60, nO, lsO]phos-phoryl group transfer catalyzed by seven phosphotransferases were simultaneously determined in the author s laboratory and in the laboratory of J.R. Knowles. The first to be completed was the demonstration of inversion by adenylate kinase however, prior to that glycerokinase, hexokinase and pyruvate kinase had been shown to catalyze [l80]thiophosphoryl group transfer with the same stereochemical consequences, either all with inversion or all with retention. Glycerokinase was later shown to catalyze both [l60, nO, l80]phosphoryl and [l80]thiophosphoryl group... 

Fig. 4. Proposed mechanism of pyruvate kinase in A, the phosphoryl transfer reaction and B, the enolization of pyruvate, based on nuclear relaxation (75, 43, 45—48) and chemical modification studies (49—51)...

Since metal coordination or immobilization of the transferred phosphoryl group by multiple hydrogen bonds would inhibit the formation of a metaphosphate intermediate in an S l mechanism and would facilitate nucleophilic attack in an Sy2 mechanism, the latter process seems likely for the reactions catalyzed by staphylococcal nuclease, DNA polymerase, pyruvate kinase, fructose diphosphatase, phosphoglucomutase, (Na + K) ATPase and possibly PEP carboxylase. In creatine kinase where an S l mechanism is possible, the enzyme would have to prevent access of nucleophiles other than ADP and creatine to the reactive metaphosphate intermediate. 

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