Studies on the Enzyme Enolase

The equilibrium constant (Kint) for the enzyme-bound substrate and product of a one substrate/one product enzyme (enolase) and for those of a two substrate/two product enzyme (creatine kinase) have been determined. The values of Kint were determined by the rapid quenching of equilibrium mixtures of enzyme and radiolabeled substrate and product, under conditions where all of the marker substrate and product are bound. The scope and limitations of this method are discussed. Values of Kint have been collected from the literature, and it is shown that these data are consistent with the theory for kinetically optimized enzymes that is developed in the preceding paper.

Section: Resultssupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase

Burbaum¹,

Knowles²

1989

“…The absolute requirement for Mg3'*' ions is clearly demonstrated, with an optimum Mg concentration near 10_s M. From the data in Figure 5 4: The effect of pH and magnesium ions on the rate of the enolase reaction. The observed rates in terms of optical density change were corrected for the effect of pH and Mg on the absorbance of enolpyruvic acid phosphate (Wold and Ballou, 1957). The buffer in both experiments was 0.05 m imidazole containing 0.2 m KC1.…”

Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of Enolase from Rainbow Trout (Salmo gairdnerii gairdnerii)^*

Cory¹,

Wold²

1966

Self Cite

Enolase has been purified and crystallized from the white skeletal muscle of rainbow trout. The crystalline enzyme contains three electrophoretically distinct enolases, and since the same ratio of these three enzyme forms was present in the original muscle extract, it is concluded that the three forms are not artifacts of isolation. Working with the mixture of the three forms, the trout muscle enolase has been characterized with respect to physical, chemical, and bio-T A he skeletal muscle of several species of Salmonidae has been reported to contain multiple forms of the common glycolytic enzyme enolase(2-phospho-D-glycerate-hydro-lyase (4.2.1.11)) (Tsuyuki and Wold, 1964). In an attempt to investigate this phenomenon in some detail in terms of both the structural and functional significance of the different forms of enolase, a pure preparation of enolase must first be obtained. This paper reports the purification and crystallization of enolase from the white skeletal muscle of rainbow trout (Salmo gairdnerii gairdnerii) and some preliminary data on the characterization of the enzyme. The three forms of the enzyme which can be detected in the original muscle extract (Tsuyuki and Wold, 1964) are present in the crystalline enzyme preparation in what appears to be unchanged proportions, and the biochemical, chemical, and physical properties reported in this paper are determined on the mixture of the three forms. Experimental ProcedureAssays. Enolase activity was determined by the direct spectrophotometric method of Warburg and Christian (1942), according to published procedures (Holt and Wold, 1961) using a Zeiss or a recording Gilford spectrophotometer. Protein determinations were based on the 280-m/i absorbance and related to accurate dry weight of the pure enzyme.The barium salt of the substrate (D-glyceric acid 2phosphate, Sigma Chemical Co.

“…Table I also shows the ratios predicted for a first-order approach to equilibrium with an equilibrium constant of 99. The amount of c(THN)AD arising from the hydrate remained Wold and Ballou (1957). 'Krebs (1953). "…”

Section: Methodsmentioning

confidence: 99%

Equilibrium of 5,6-hydration of NADH and mechanism of ATP-dependent dehydration

Acheson¹,

Kirkman²,

Wolfenden³

1988

At equilibrium, water addition to the 5,6 double bond of NADH was observed to favor the hydrate by a factor of approximately 100. Hydration generates two epimers of NADHX (beta-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide). Only the 6S epimer of the hydrate was found to serve as a true substrate for an ATP-dependent dehydratase from yeast that regenerates NADH. Yet enzymatic conversion of both epimers of the hydrate to NADH was found to proceed essentially to completion in the presence of ATP and dehydratase. This is explained by the observed ability of the epimers to undergo rapid spontaneous equilibration, so that it is unnecessary to postulate a lack of stereospecificity in the dehydratase.