title: Cryoenzymic studies on yeast 3-phosphoglycerate kinase. Attempt to obtain the kinetics of the hinge-bending motion.
abstract: This is a continuation of a study on the 3-phosphoglycerate kinase (PGK) reaction in the direction of 1,3-bisphosphoglycerate (bPG) formation: ATP + 3-phosphoglycerate (PG) <==> ADP + bPG [Schmidt, P. P., Travers, F., & Barman, T. (1995) Biochemistry 34, 824-832]. We showed that species containing bPG accumulate in the steady state, but their low concentrations and rapid kinetics of formation precluded a full study, even under cryoenzymic conditions in 40% ethylene glycol. Here we studied the PGK reaction in 30% methanol. The transient kinetics of bPG formation were obtained by chemical sampling: PGK was mixed with PG and [gamma-32P]ATP in a rapid flow quench apparatus, the mixture aged 4 ms up and quenched in acid, and the [1-(32)P]bPG was determined. The time course consisted of a rapid rise of bPG (kinetics k(obs)) and a steady state phase. In methanol, the amplitude of the rise was large (>50% of the PGK in the steady state), and k(obs) was measurable. Fluorescence stopped flow was used to study the formation of the binary E x PG and E x ATP. The affinities of PGK for ATP and PG were high in methanol (Kd = 102 and 1.5 microM, respectively), but the kinetics of the formation of E x PG and E x ATP were too rapid to be measured. From these and the chemical sampling experiments, we propose a reaction scheme for PGK: a rapid formation of the collision complex E x PG x ATP (K1), a slow isomerisation to E* x PG x ATP (k2,k(-2)), a rapid phosphorylation transfer step to E x bPG x ADP (K3), and a slow release of the products (k4). In our scheme, k(obs) is the reflection mainly of k2 and k(-2) and the steady state of k4. Using a computer simulation procedure, k2/K1 = 0.37 microM(-1) s(-1), k(-2) = 33 s(-1), K3 = 4, and k4 = 7.1 s(-1). We propose that k(obs) measures the kinetics of the putative hinge-bending motion of PGK, i.e., the conformational change that is necessary for the substrates to line up for phosphoryl transfer.
authors.affiliation: INSERM U128, CNRS, BP 5051, Montpellier, France.
authors.forename: A
authors.initials: A
authors.lastname: Geerlof
authors.forename: P P
authors.initials: PP
authors.lastname: Schmidt
authors.forename: F
authors.initials: F
authors.lastname: Travers
authors.forename: T
authors.initials: T
authors.lastname: Barman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005615:Freezing; D005656:Fungal Proteins / Q000378:metabolism*; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012997:Solvents; D013050:Spectrometry, Fluorescence
chemical_list: D004163:Diphosphoglyceric Acids; D005656:Fungal Proteins; D005988:Glyceric Acids; D012997:Solvents; C015891:glycerate 1,3-biphosphate; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D000432:Methanol