Sulfhydryl groups of yeast phosphofructokinase-specific localization on beta subunits of fructose 6-phosphate binding sites as demonstrated by a differential chemical labeling study.

M.N Tijane,A.F Chaffotte,F.J Seydoux,C Roucous,M Laurent

doi:10.1016/s0021-9258(19)70446-3

Abstract

Yeast phosphofructokinase contains 83 +/- 2 cysteinyl residues/enzyme oligomer. On the basis of their reactivity toward 5,5-dithiobis(2-nitrobenzoic acid), the accessible cysteinyl residues of the native enzyme may be classified into three groups. For titrations performed with N-ethylmaleimide, subdivisional classes of reactivity are evidenced. In each case, the 6 to 8 most reactive cysteines are not protected by fructose 6-phosphate from chemical labeling and do not seem involved in subsequent enzyme inactivation. Differential labeling studies as well as direct protection experiments in the presence of fructose 6-phosphate, indicate that 12 -SH groups/enzyme oligomer (i.e. three -SH groups per binding site) are protected by the allosteric substrate from the chemical modification. Specific labeling by the differential method of the cysteinyl residues protected by fructose 6-phosphate and further separation of the two types of subunits constituting yeast phosphofructokinase, show that the substrate binding sites are localized exclusively on subunits of beta type. Thus, alpha subunits are not implicated directly in the catalytic mechanism of yeast phosphofructokinase reaction.

Highlights

Yeast phosphofructokinase contains83 k 2 cysteinyl existence of a small number of interacting residues/enzyme oligomer
Differential labeling studies as well as direct protection experimentsin the presence of fructose 6-phosphate, indicate that 12 ”SH groups/enzyme oligomer (Le. three ”SH groups per binding site) are protected by the allosteric subedof the sulfhydryl groups of the protein and we study the protecting effect of fructose 6-phosphate on the chemical modification of the enzyme
The numberof residual sulfhydryl groups of native phosphofructokinase withunlabeled MalNEt in the which had not reacted with MalNEt was determined under presence of 5 m~ fructose 6-phosphate,the protecting ligand conditions (8M urea) which have been shown to denature the and theexcess of unlabeled reagent were eliminated by filtraenzyme and toexpose all of its " S H groups to reaction with tion on a Sephadex G-50 column

Summary

RESULTS

Centration was determined by titration with an excess of dithiothreitol. The increase of absorbance was measured a t 412 nm and 25°C on a Cary model 16 recording spectrophotometer and the number of “ S H groups titrated was calculated using a molar extinction coefii-. The reaction mixture was filtered on the small Sephadex G-50 phosphofructokinase remains fully activeafter titration with column and reincubated with radioactive MalNEt or with NbSz. Separation of Yeast Phosphofructokinase Subunits under Dena- that the first class of reactive cysteines is not involved in turing Conditions-Yeast phosphofructokinase ranging from 4 to 9 mg was denatured by incubation in 25 mM Tris/sulfonic acid buffer (pH 8.6) containing 50 mM glycine, 1mM EDTA, 100 mM 2-mercapenzyme inactivation which can be related to the modification of a large numberof slowly reacting cysteines. The numberof residual sulfhydryl groups of native phosphofructokinase withunlabeled MalNEt in the which had not reacted with MalNEt was determined under presence of 5 m~ fructose 6-phosphate,the protecting ligand conditions (8M urea) which have been shown to denature the and theexcess of unlabeled reagent were eliminated by filtraenzyme and toexpose all of its " S H groups to reaction with tion on a Sephadex G-50 column. 0.026mk" and b parameter (see text) was equal to 0.267

FRACTION NUMBER

Findings

DISCUSSION