Purification, kinetic studies, and homology model of Escherichia coli fructose-1,6-bisphosphatase

doi:10.1016/S0167-4838(01)00261-8

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1594, Issue 1, 31 January 2002, Pages 6-16

Biochimica et Biophy...

https://doi.org/10.1016/S0167-4838(01)00261-8 Get rights and content

Abstract

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The k_cat of the E. coli FBPase was 14.6 s⁻¹ as compared to 21 s⁻¹ for the pig kidney enzyme, while the K_m of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg²⁺ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg²⁺, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg²⁺ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.

Introduction

Fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) catalyzes a control step in the gluconeogenic pathway, specifically, the hydrolysis of fructose-1,6-bisphosphate (Fru-1,6-P₂) to fructose-6-phosphate (Fru-6-P) and P_i [1]. FBPases have been isolated from many sources including human liver, pig kidney, rabbit liver, beef liver, and Escherichia coli [2]. The investigation of their structure and function has led to a better understanding of their mechanism of action. The amino acid sequences of mammalian enzymes are 85% identical to each other. In addition, the amino acid sequence of the enzyme from E. coli is 42% identical and 55% similar to the sequence of the pig kidney enzyme, which implies a similar structure. FBPases are multimeric enzymes and in most cases are tetramers composed of four identical subunits [3], [4]. In previous studies, the E. coli enzyme at low concentrations was shown to be a tetramer by gel filtration [5].

Most FBPases exhibit Michaelis-Menten kinetics at physiological pH and require a divalent metal cation, such as Mg²⁺, for activity. For the mammalian enzymes the catalytic activity of FBPase is inhibited by AMP, which causes the enzyme to undergo a transition from a high-activity R (Relaxed) state to an inactive T (Tense) state [6]. For the mammalian enzymes, AMP inhibits cooperatively with a Hill coefficient near 2 [7], [8]; however, cooperative binding of AMP to E. coli FBPase has not been reported. Mammalian FBPases are also competitively inhibited by fructose-2,6-bisphosphate (Fru-2,6-P₂), with a K_i of 0.065 μM reported for the pig kidney enzyme [9]; however, previous studies on the E. coli enzyme showed varying results for this inhibitor. One study showed that Fru-2,6-P₂ did not inhibit E. coli FBPase [5] while another reported that Fru-2,6-P₂ was a competitive inhibitor [10]. E. coli FBPase has not been successfully purified to homogeneity, with previous characterization performed at an estimated purity level of only about 50% of total protein [5], [10], [11]. By cloning the E. coli fbp gene downstream from the strong T7 promoter on a high copy number plasmid we have over-expressed the enzyme. The higher expression levels have allowed the development of a new purification of E. coli FBPase from total protein to 98% purity. The quaternary structure and kinetic properties of this purified material were investigated and a homology model for the structure of the E. coli enzyme was constructed based upon the available three-dimensional structure of the pig kidney enzyme. The resulting model was analyzed in view of the kinetic properties determined for the E. coli enzyme as compared to those found for the pig kidney FBPase. Understanding the differences between the E. coli and pig kidney FBPase may help to elucidate the mechanism of the allosteric signal transmission in these FBPases.

Section snippets

Materials

Unless otherwise indicated, all components used in culture media, buffers and kinetic measurements were purchased from Sigma Chemical Co. Tris and enzyme-grade (NH₄)₂SO₄ were from ICN Biomedicals. Yeast extract, tryptone and casamino acids were from Difco. AEBSF was from Calbiochem. The reagents for DNA sequencing, restriction endonucleases, T4 DNA ligase, T4 DNA polymerase, and T4 polynucleotide kinase were from Amersham Pharmacia Biotech and New England Biolabs, and were used according to the

Results and discussion

The study of E. coli FBPase has been hindered by the inability to prepare the enzyme in pure form [5]. Contradictory kinetic properties of partially purified E. coli FBPase with purity levels of about 50% of total protein have been reported [5], [10]. In this investigation, a purification scheme for E. coli FBPase has been devised and a purity level as high as 98% has been attained. Also, a homology model was created in order to compare the E. coli FBPase to the pig kidney enzyme, the structure

Acknowledgements

This work was supported by a grant from the American Diabetes Association.

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Purification, kinetic studies, and homology model of Escherichia coli fructose-1,6-bisphosphatase

Abstract

Introduction

Section snippets

Materials

Results and discussion

Acknowledgements

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