Substrate-Assisted Movement of the Catalytic Lys 215 during Domain Closure:  Site-Directed Mutagenesis Studies of Human 3-Phosphoglycerate Kinase

Abstract

3-Phosphoglycerate kinase (PGK) is a two-domain hinge-bending enzyme. It is still unclear how the geometry of the active site is formed during domain closure and how the catalytic residues are brought into the optimal position for the reaction. Comparison of the three-dimensional structures in various open and closed conformations suggests a large (10 A) movement of Lys 215 during domain closure. This change would be required for direct participation of this side chain in both the catalyzed phospho transfer and the special anion-caused activation. To test the multiple roles of Lys 215, two mutants (K215A and K215R) were constructed from human PGK and characterized in enzyme kinetic and substrate binding studies. For comparison, mutants (R38A and R38K) of the known essential residue, Arg 38, were also produced. Drastic decreases (1500- and 500-fold, respectively), as in the case of R38A, were observed in the kcat values of mutants K215A and K215R, approving the essential catalytic role of Lys 215. In contrast, the R38K mutation caused an only 1.5-fold decrease in activity. This emphasizes the importance of a very precise positioning of Lys 215 in the active site, in addition to its positive charge. The side chain of Lys 215 is also responsible for the substrate and anion-dependent activation, since these properties are abolished upon mutation. Among the kinetic constants mainly the Km values of MgATP and 1,3-BPG are increased (approximately 20- and approximately 8-fold, respectively) in the case of the neutral K215A mutant, evidence of the interaction of Lys 215 with the transferring phospho group in the functioning complex. Weakening of MgATP binding (a moderate increase in Kd), but not of MgADP binding, upon mutation indicates an initial weak interaction of Lys 215 with the gamma-phosphate already in the nonfunctioning open conformation. Thus, during domain closure, Lys 215 possibly moves together with the transferring phosphate; meanwhile, this group is being positioned properly for catalysis.

An Allosteric Signaling Pathway of Human 3-Phosphoglycerate Kinase from Force Distribution Analysis

“…Our allosteric pathway overlaps with a previously identified transmission path, which however was restricted to a single hinge in sheet L [18]. While previously identified key residues involved in PGK domain closure [1], [18], [34], [36] are part of this pathway, underlining their functional relevance, residues Gly371, Gly372 and Gly394 are residues newly uncovered as critical allosteric spots. We hypothesize that an addition of a sidechain to these residues would abolish or measurably alter hPGK allostery, and thus suggest them as interesting candidates for future mutational studies.…”

“…By comparing the absolute force magnitudes for the substrates, we observe that BPG exerts stronger forces on its binding residues. According to experimental binding measurements, BPG also shows a higher binding affinity for hPGK as compared to ADP [34]. Thus, here, the higher BPG-hPGK interaction forces, which are mostly attractive and of electrostatic nature, reflect steeper binding potentials, i.e.…”

Terazosin activates Pgk1 and Hsp90 to promote stress resistance

“…We rationalize that, based on the chemical equilibrium principle, the effective TZ dosage, for either inhibition or activation, should depend on the relative affinity and the concentration of TZ, ADP and ATP for Pgk1. The Pgk1-binding affinity (K d ) of ADP and ATP is 2.9×10 -5 M and 3.3×10 -4 M, respectively 24 . According to our measurement by ITC (Isothermal Titration Calorimetry), TZ and Ta bind to Pgk1 with the affinity (K d ) of 2.78×10 -6 M and 2.5×10 -6 M, respectively (Supplementary Fig.…”

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

“…SAXS Experiments-Expression, purification, and activity measurements of recombinant wild type HsPGK were performed as described previously (18,22). Small angle x-ray scattering data were collected at the bioSAXS beamline ID14-3 (23) at the European Synchrotron Radiation Facility (Grenoble, France) with a Pilatus 1M detector (Dectris Ltd., Baden, Switzerland) at a wavelength of 0.931 Å and a camera length of 2.42 m. Scattering curves were measured from solutions of HsPGK without substrates; the binary complexes with either 50 mM 3PG or 10 mM ADP; a quaternary complex inhibited with the TSA 3PG-AlF 4 Ϫ -ADP (50 mM 3PG, 10 mM ADP, 25 mM MgCl 2 ,10 mM NH 4 F, and 2 mM AlCl 3 added 3 h before measurements); the 3PG-ADP ternary complex (50 mM 3PG, 10 mM ADP, and 25 mM MgCl 2 ) and the 3PG-ATP ternary complex (50 mM 3PG, 10 mM ATP, and 25 mM MgCl 2 , nucleotide added just before measurements) in SAXS buffer (50 mM Tris, pH 7.5, and 20 mM DTT).…”

“…The adenosine moiety is bound in a similar manner to the ADP ternary complex, but the ␤-and ␥-phosphate groups protrude into the solvent and are poorly ordered: A conformation that is similar to the same complex of pig muscle PGK (40). As the ␥-phosphate has been shown to possess a high extent of mobility, it could temporarily interact with Lys 215 , even in the open conformation (22). Thus, ATP may be able to stabilize the conformation seen in the HsPGK-3PG-ADP-open structure, which is similar to that observed in the closed conformation (3PG-MgF 3 Ϫ -ADP TSA complex) with Lys 215 interacting with the phosphate groups of 3PG and the transition state analogue (Fig.…”