Identification of interactions that stabilize the transition state in Escherichia coli phosphofructo-1-kinase

R.L Zheng,R.G Kemp

doi:10.1016/s0021-9258(17)32333-5

Abstract

The activity of Escherichia coli phosphofructo-1-kinase depends upon the dissociation of a group with a pK of approximately 6.6. Mutation of the 2 active site residues most likely to be titrated in this range, Asp-127 and Asp-129, lowered activity but produced little change in the pH dependence, suggesting that these residues while important to activity were not responsible for the pH dependence of the enzyme. Alternatively, the pH dependence was thought to correspond to the negative charge on the phosphoryl group being transferred in the reaction. Mutation of Arg-72 to histidine, while lowering the activity substantially, shifted the pH optimum to approximately 6.2 with a secondary plateau on the alkaline limb that was eliminated in the double mutant R72H/R171H. Mutation of Arg-171 alone produced only a small decrease in maximal activity and little change in pH dependence from the wild type enzyme. The data support an associative mechanism with a transition state stabilized by the interaction between the negative charge on the phosphoryl group being transferred and the arginyl residue at position 72 on the enzyme.

Highlights

The activity of Escherichia coli phosphofructo-l-ki- 129 and concludedthat t h e Asp-127 is not directly involved in nase depends upon thdeissociationof a group with pKa the pH dependenceof the enzymeand that a mutation of Aspof approximately 6.6
We suggest that the pH dependence,suggestingthat these residues the pH dependente reflectsthe pK of a phosphoryl groupof ATP
ThpeH de- enzyme.,single and doublemutantsinvolving pendence was thought to correspond to the negative basic residues reputed to be presenitn the active site of phoscharge on the phosphoryl groubpeing transferred in the phofructo-1-kinase, Arg-72 a n d Arg-171, have been studied to rtpheraeocxaticimotniav.tietM ylysuu6tba.2sttioawnnotiftiahAlrlyga,7ss2ehtciofotnhedidsatritdhyiepnHpel,aowtpehtaiimuleuolmonwtteohreinapga-lktiadraelinnnsteiiftyiotnhestraetsei.due that is critical to charge stabilizatioinnthe limb that was eliminated in the double mutant R72W R171H

Summary

Introduction

The activity of Escherichia coli phosphofructo-l-ki- 129 and concludedthat t h e Asp-127 is not directly involved in nase depends upon thdeissociationof a group with pKa the pH dependenceof the enzymeand that a mutation of Aspof approximately 6.6. Mutation okf g-171alone produced only a small decrease in maximal activity and little change in pH Strains-E. coli strain DF1020(pro-82, Dpfi[201], recA56, D(rhadependence from the wild type enzyme.

Results

Conclusion