Stopped Flow Spectrophotometric Studies of Yeast Enolase Subunit Interaction

John M Brewer,Richard J Desa

doi:10.1016/s0021-9258(20)81792-x

Abstract

Abstract We measured the kinetics of absorbance changes at 296 nm occurring on addition to or removal of magnesium from brewers' and bakers' yeast enolase A, B, or a mixture of these. The stopped flow apparatus was equipped with a highly stabilized Xenon arc light source and an on-line computer was used to record and process all data. When excess magnesium is added to partially dissociated enolase, we obtain a rapid absorbance change within the dead time of the instrument, a protein concentration-dependent change, and finally a first order change. The initial absorbance change is probably the result of magnesium binding to both monomeric and dimeric enzyme. The protein concentration-dependent change is from subunit association. The final change parallels recovery of enzymatic activity. The rates of these reactions are slower in 1 m potassium chloride, which evidently reduces the rate of subunit association. The source of the enolase, the isozymic form of the enzyme, and the age and storage conditions determine how readily the enzyme dissociates in the absence of metal. The initial activity of enzyme is inversely proportional to the absorbance changes observed in the stopped flow, that is, to the extent of dissociation. Associated enzyme is fully active within 4 ms of binding magnesium, suggesting that magnesium binding to yeast enolase monomers is not requisite for subunit association. On addition of EDTA to enzyme containing endogenous magnesium, two successive first order reactions occur, following an absorbance change within the dead time of the instrument. This occurs whether the enzyme dissociates under these conditions or not, showing that these transitions do not correspond to those seen on addition of magnesium. Consequently, at least four transitions occur in the enzyme on removal of magnesium. We conclude that magnesium can affect the subunit association equilibrium simply by binding preferentially to the dimeric form of enolase.

Highlights

The stopped flow apparatus was equipped with a highly stabilized Xenon arc light source and an on-line computer was used to record and process all data
The stopped flow instrument waskept at 20” with a circulating pump and a AbsorbanceChanges Produced by Magnesium Addition to En&se-Addition of excess+magnesiumto a mixture of brewers’yeast enolaseA, B, etc., in 3 mM EDTA and either 1 M potassium chloride, 1 M potassium acetate, or buffer alone results in changesin tryptophan absorbance
We should plot only the reciprocal of the absorbance change due to the second order part of the reaction, but we find it difficult to separate this phase from the rest of the reaction

Summary

Methods

Enolase was purified from brewers’ or bakers’ yeast by the method of Westhead and McLain [10]. Brewers’ yeast was a generous gift of Carlings, Inc., of Atlanta, and bakers’ yeast was purchased from a local food wholesaler. The specific activities of our preparations of the enzyme were equal to or greater than the literature values [10]. The preparations consist of a mixture of forms of enolase, called A, B, C, etc., in order of their elution from a triethylaminoethylcellulose column [10]. Our preparations are largely the A and B forms. We separated these by chromatography on TEAEr-cellulose as described by Westhead and McLain (lo), except we used 5 times the gradient volume used by those authors [3].

Results

Discussion

Conclusion