The Steady‐State Kinetics of Yeast Phosphoglycerate Kinase

Abstract

1.A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bisphosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used.2. Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation.3. The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration.4. The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5. Salt activation is steepest when the substrate concentrations are low ; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects.6. The presence of 20 mM or more Na2S04 converted non-linear kinetic plots to linear ones.

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

“…At low concentrations of fixed substrate ( 1 mM), the isolated bacterial PGK enzymes exhibited the non-linear kinetics (using an Eadie-Hoffstee plot) common for this enzyme [12]. To minimize this phenomenon, the concentration of the fixed substrate was increased.…”

“…PGK has been purified from a wide variety of sources [1][2][3], and its conserved three-dimensional structure is known [4][5][6][7][8]. Detailed kinetic and mechanistic investigations have been conducted with the yeast enzyme [9][10][11][12][13][14][15], and most aspects of its kinetic behaviour are understood. However, one complicating factor in using PGK is the occurrence of non-linear kinetics (apparent negative co-operativity) exhibited when PGK is assayed in the reverse direction to glycolysis (the 3-phosphoglyceric acid [PGA] to 1,3-bisPGA direction).…”

“…One suggested explanation is multiple binding sites for each substrate [16][17][18], although the crystallographic structure does not directly support this. Another explanation put forward is slow product dissociation at low salt\substrate concentrations [12]. However, linear kinetics are restored by the presence of sulphate or other anions.…”

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase

“…Enolase is thus purified at the first step by differential Scopes, 1978). Its specific activity is of the same order the first dye-ligand adsorbent is not important, provided (700-(900 units/mg), although high concentrations of that it binds at least 65%, and preferably 75%, of the substrates were not necessary to obtain such values.…”

“…Its protein in the stated conditions. We have also used kinetic properties do differ, in that there is a much less Procion Green HE-4BD and Remazol Brilliant Blue R as pronounced substrate-activation effect (Scopes, 1978) well as the two mentioned in the procedure above.…”