Chemical cross-linking stabilizes the enzymic activity and quaternary structure of formyltetrahydrofolate synthetase.

M De Renobales,W Welch

doi:10.1016/s0021-9258(19)70486-4

Abstract

Clostridium cylindrosporum formyltetrahydrofolate synthetase tetramers cross-linked with dimethyl suberimidate remained active in the absence of the monovalent cations normally required for enzymic activity and the tetrameric conformation. The modified enzyme was analyzed by sodium dodecyl sulfate electrophoresis, sedimentation velocity, and gel permeation chromatography. Under the experimental conditions used, the enzyme was only partially cross-linked; 74% of the enzyme was cross-linked dimer or monomer. Nonetheless, the modified enzyme is able to retain enzymic activity and the tetrameric structure under conditions where native enzyme would be completely dissociated and inactivated. The result suggests that cross-linked dimers strongly associate with each other and with monomers. Flame emission spectroscopy indicates that cross-linked enzyme contains two monovalent cations per tetramer.

Highlights

EXPERIMENTALPROCEDURES the enzymewas only partiallycross-linked;74% of the Unless otherwise noted, reagents were ACS reagent grade or better. enzyme was cross-linked dimer or monomer
Flameemission spectroscopy indicates that cross-linked enzyme containstwo monovalent cations routinely diluted inice-cold0.05 M potassium maleate, 0.1 M 2
Like many other enzymes (l),the enzyme isolated from Clostridium cylindrosporum requiretshe presence of monovalent cationsfor optimal activity [2,3,4,5,6].The active formof the enzyme is a tetramer

Summary

RESULTS

Gel pattern which is similar to that observed for many other proteins [20].Electrophoresis of control (unmodified)enzyme. The mobilities were consistent imidate cross-linking of formyltetrahydrofolate synthetase on from experiment to experiment and gave the expected logathe kinetic properties of the enzyme. The mobility cation caused a drop in specificactivity of between 50 and 80% of monomer derived from either native or cross-linked protein (see Tables I and 11). a large fraction of the enzymic was identical. 0.3 mM, Kf,,., = 4.9 mM) nor Wac, the stimulation by mono- at the top of the gel.

Stirnuactivitv lation

FRACTICN NUMBER

Findings

DISCUSSION