Direct Kinetic Evidence That Lysine 215 Is Involved in the Phospho-Transfer Step of Human 3-Phosphoglycerate Kinase

Abstract

3-Phosphoglycerate kinase (PGK) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. PGK is a key enzyme in glycolysis; it catalyzes the reversible reaction 1,3-bisphosphoglycerate + ADP <--> 3-phosphoglycerate + ATP. Here we explored the catalytic role in human PGK of the highly conserved Lys 215 that has been proposed to be essential for PGK function by a transient and equilibrium kinetic study with the active site mutant K215A. By the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type PGK and mutant K215A. By the use of a chemical sampling method (rapid quench flow) under multiple and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild-type PGK follows product formation (presumably product release), whereas with the mutant it is the phospho-transfer step itself that is rate-limiting. Mutant K215A has a low inherent phosphotransferase activity, and to explain this, we carried out a molecular modeling study. This suggests that with the mutant the conserved Arg 65 replaces the missing Lys 215 by helping to position the transferable phospho group during the reaction. Molecular dynamics simulations suggest that in the mutant the closed conformation of the enzyme is stabilized by a salt bridge between Asp 218 and Arg 170 rather than Arg 65 in the wild-type PGK.

Two distinct glyceraldehyde‐3‐phosphate dehydrogenases in glycolysis and gluconeogenesis in the archaeon Haloferax volcanii

“…The recombinant enzyme was characterised as 45 kDa monomer and is thus similar to most characterised PGKs from Bacteria, Eukarya and Archaea. Characterised bacterial and eukaryal PGKs include those from Thermotoga maritima and Homo sapiens . PGKs from Bacteria and Eukarya are amphibolic enzymes involved in both glucose catabolism and gluconeogenesis.…”

Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: implications for the design of better anti-HIV analogues

“…Yet, the initial interactions with substrates are always very crucial in respect to the further elementary steps of catalysis, as indicated by fast kinetic studies with hPGK as an example. 32,48,49 Since each kinase has its own mechanism for the binding and the phosphorylation of nucleotides, probably with a different time-course from kinase to kinase, using molecular dynamics simulation it would have been extremely difficult and timeconsuming to carry out the experiments on the appropriate (different) time-scale in each case. Therefore, here we restricted ourselves to do simple docking studies with the known active (closed) conformational states of these kinases and in each case we used energy minimization after the docking steps.…”

Interaction of Human 3-Phosphoglycerate Kinase with Its Two Substrates: Is Substrate Antagonism a Kinetic Advantage?

“…34 In that work, the experiments were limited to the concentration range 10-60 μM D-ADP. Here, we extended the range to 100 μM D-ADP.…”

“…It is a point-by-point method, and the time course of product formation is obtained from several experiments. Here, with PGK, we measured ATP formation by HPLC as in the work of Varga et al 34 This method is specific for ATP, but because intermediates containing ATP break down in the acid quench, the ATP measured represents PGK-bound ATP and free ATP. Two apparatuses were used: an in-house development 34 and QFM-400 from Bio-Logic (Claix, France).…”

“…Here, with PGK, we measured ATP formation by HPLC as in the work of Varga et al 34 This method is specific for ATP, but because intermediates containing ATP break down in the acid quench, the ATP measured represents PGK-bound ATP and free ATP. Two apparatuses were used: an in-house development 34 and QFM-400 from Bio-Logic (Claix, France). With the inhouse apparatus, the time courses can be measured from 3 ms to 300 ms; with the Bio-Logic apparatus, the time courses can be measured from 6 ms to several seconds.…”