Nucleotide Binding to Pig Muscle 3-Phosphoglycerate Kinase in the Crystal and in Solution:  Relationship between Substrate Antagonism and Interdomain Communication

Abstract

Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, beta,gamma-methyleneadenosine-5'-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2'3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the K(d) values for MgADP were found to be 0.096 +/- 0.021 and 0.045 +/- 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both K(d) values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 +/- 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 +/- 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg(2+)-free) binding (K(d) = 0.34 +/- 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg(2+) ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg(2+) binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.

Magnetic isotope effect of magnesium in phosphoglycerate kinase phosphorylation

“…Thus, our current understanding of ATP-synthesizing enzymes is still rudimentary at the molecular level and the key reaction responsible for the formation of the energy-carrying chemical bond POOOP remains obscure. Indeed, the structures of the protein parts and catalytic sites of phosphorylating enzymes, such as ATP synthase, creatine kinase, and phosphoglycerate kinase (PGK) (2)(3)(4)(5)(6)(7)(8)(9)(10), are well known. There is an understanding of their functioning as molecular rotary motors (referring to ATP synthase) or molecular pumps (creatine kinase, for instance); however, within the area of enzymatic reaction chemistry, all ideas are limited to speculations circulating mostly around nucleophilic mechanisms.…”

Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates

“…This disturbs the coordination of the metal ion and its linkage with ␣-, ␤-, and ␥-phosphates and results in interaction of metal ion with Asp 375 and ␣-and ␤-phosphates. This leads to the collapse of the transition state to form metal-ADP and 1,3-biphosphoglycerate complexes bound to PGK, which then reverses the conformational change and releases the products (27)(28)(29). As with any reversible enzyme it is generally assumed that the phosphorylation of ADP to ATP using 1,3-biphosphoglycerate as a phosphate donor would also go through a similar transitional state (ED* ϭ EP*).…”

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

“…This effect is called substrate antagonism. [2][3][4] With the phosphotransferase group of enzymes, both substrate synergy and substrate antagonism have been observed. For example, yeast hexokinase 5 and rabbit muscle creatine kinase 6 are subject to substrate synergy, whereas phosphofructokinase from Escherichia coli, 2,7 Rho-associated protein kinase (ROCK I; an enzyme that is implicated in cell adhesion and smooth muscle adhesion), 8 and, in particular, 3-phosphoglycerate kinase (PGK; EC 2.7.2.3) 9,10 are subject to substrate antagonism.…”

“…22 Recently, Cliff et al reported a closed structure of hPGK. 11 High-resolution structures of the transition-state analogue complexes PGK·PG·MgF 3 − ·ADP and PGK·PG·AlF 4 − ·ADP, where metal fluorides replace the transferable phosphate of ATP, were obtained. It was suggested that a zero local net charge at the active site is a key feature of the phosphotransfer process.…”

“…Thus, K m /K d N 1 with PG 4,10,24 and with both ATP and ADP. 3 Merli et al provided evidence that antagonism is connected to the domain closure induced by the substrates. In a review of the work of Bernstein et al 19 on the closed structure of PGK from T. brucei, Blake 25 proposed that the small conformational change caused by PG binding alone "primes" the enzyme to react differently from the ligand-free enzyme to the binding of ATP and ADP, and that the heart of this priming mechanism is helix 14, which belongs to the C-terminal end of hPGK but is located in the N-domain.…”