Structure of the R65Q Mutant of Yeast 3-Phosphoglycerate Kinase Complexed with Mg-AMP-PNP and 3-Phospho-d-glycerate^,

Abstract

The structure of a ternary complex of the R65Q mutant of yeast 3-phosphoglycerate kinase (PGK) with magnesium 5'-adenylylimidodiphosphate (Mg-AMP-PNP) and 3-phospho-D-glycerate (3-PG) has been determined by X-ray crystallography to 2.4 angstrom resolution. The structure was solved by single isomorphous replacement, anamalous scattering, and solvent flattening and has been refined to an R-factor of 0.185, with rms deviations from ideal bond distance and angles of 0.009 angstrom and 1.78 degrees, respectively. PGK consists of two domains, with the 3-PG bound to a "basic patch" of residues from the N-terminal domain and the Mg-AMP-PNP interacting with residues from the C-terminal domain. The two ligands are separated by approximately 11 angstrom across the interdomain cleft. The model of the R65Q mutant of yeast PGK is very similar to the structures of PGK isolated from horse, pig, and Bacillus stearothermophilus (rms deviations between equivalent alpha-carbons in the individual domains < 1.0 angstrom) but exhibits substantial variations with a previously reported yeast structure (rms deviations between equivalent alpha-carbons in the individual domains of 2.9-3.2 angstrom). The most significant tertiary structural differences among the yeast R65Q, equine, porcine, and B. stearothermophilus PGK structures occur in the relative orientations of the two domains. However, the relationships between the observed conformations of PGK are inconsistent with a "hinge-bending" behavior that would close the interdomain cleft. It is proposed that the available structural and biochemical data on PGK may indicate that the basic patch primarily represents the site of anion activation and not the catalytically active binding site for 3-PG.

Role of Phosphate Chain Mobility of MgATP in Completing the 3-Phosphoglycerate Kinase Catalytic Site: Binding, Kinetic, and Crystallographic Studies with ATP and MgATP

“…However, the conformation of the phosphate chain and location of the phosphates of AMP-PNP and AMP-PCP are completely different. While that of AMP-PNP resembles the location of ADP phosphates close to the N-terminus of helix 13, here without involvement of Mg 2+ (13,17,18), the phosphates of AMP-PCP bind to an alternative site at the N-terminus of helix 8, through the metal ion (20). Solution of the complete NMR structures of both the bound MnADP and MnATP (45) has not solved the above uncertainties.…”

“…(i) How do the orientation and interactions of phosphates of bound MgATP relate to those extremely different orientations and interactions observed in the previous crystal structures with bound Mg(Mn)AMP-PNP (13,17,18) and with MgAMP-PCP (20)? (ii) Is there any special role for the metal ion, in addition to the generally expected ones, in formation of the catalytic complex of PGK with each nucleotide substrate?…”

Highly Reactive Cysteine Residues in Rodent Hemoglobins

“…Mammalian 3-phosphoglycerate kinase contains two thiols that react with a second order rate constant of 6.4 ϫ 10 2 to 1.1 ϫ 10 3 M Ϫ1 s Ϫ1 (5). While these cysteine residues are not directly involved in the catalytic mechanism, cysteine conjugation inhibits enzyme activity, possibly through the prevention of conformational changes required for catalysis (6,7). The fast-reacting thiols of rabbit aldolase, also not directly involved in catalysis (8), only react with a second order rate constant of 13.0 to 2.62 ϫ 10 2 M Ϫ1 s Ϫ1 (9,10).…”

Orientation of 1,3-Bisphosphoglycerate Analogs Bound to Phosphoglycerate Kinase

“…We have not identified strong requirements for functionality within the ligand, although substrate-like functionality provides weak additional affinity. Unknown, except that a crystal structure of 3-PGA bound to the R65Q mutant of yeast PGK had "a significant population" of molecules bound in the opposite orientation (8).…”

“…The C-terminal domain is the binding site for ATP/ADP. The enzyme has a number of crystal structures (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In many the two domains are in an open conformation with the substrates ϳ11 Å apart, which is much too far for direct phosphotransfer.…”