Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis

https://doi.org/10.1016/j.abb.2013.10.007Get rights and content

Highlights

  • The GAPDH from Mycobacterium tuberculosis is stabilized by NAD+, allowing for its purification for the first time.

  • The kinetic mechanism proceeds through an unusual nucleotide exchange reaction.

  • The catalytic cysteine and histidine residues have been identified using thiol alkylation studies and site-directed mutagenesis.

  • The chemical mechanism has been determined using a combination of primary, solvent and multiple kinetic isotope effects.

Abstract

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD+, then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD+ on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.

Introduction

The etiological agent of tuberculosis, Mycobacterium tuberculosis (Mtb), has infected nearly one-third of the human population [1]. Approximately 10% of TB-infections lead to an active, symptomatic infection that resulted in nearly 1.4 million deaths in 2011 [1]. In addition, multi-drug resistant strains have been reported in every country surveyed by the World Health Organization [1]. Yet some of the most basic metabolic enzymes of this bacterium have yet to be characterized.

Glyceraldehyde 3-phosphate dehydrogenase is a highly conserved enzyme that is utilized in central carbon metabolism by some of the most ancient forms of life [2]. GAPDH is best known for its role in glycolysis, catalyzing the reversible conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and NAD+ to 1,3-bisphosphoglycerate (1,3-BPG)1 and NADH [3]. This dehydrogenase is also unusual in that it utilizes a covalent thiohemiacetal intermediate to promote hydride transfer and catalysis [3]. The reaction of GAPDH is essential for the regeneration of the two molecules of ATP used to phosphorylate the hexose carbon source, glucose. The cleavage of fructose-1,6-bisphosphate yields the two triose phosphates that are interconverted into G3P. The oxidation of the aldehyde and substrate-level phosphorylation catalyzed by GAPDH generate NADH and the high energy carboxy-phosphoric anhydride containing 1,3-bisphosphoglycerate (1,3-BPG) that is used in the subsequent reaction catalyzed by 3-phosphoglycerate kinase to regenerate the two molecules of ATP used earlier in the glycolytic sequence. The very reactive nature of the product of GAPDH, 1,3-BPG, has recently been shown to be capable of non-enzymatic modification of proteins, including GAPDH [4].

Recent studies have also found GAPDH to be involved in a variety of cellular processes in addition to its major role in glycolysis. GAPDH has been shown to play a role in transcription, assisting in the formation of both DNA and RNA binding complexes as well as acting as a transcription factor co-activator [5], [6], [7]. Additionally, GAPDH has been identified as a microtubule-binding protein, a lactoferrin receptor, and as an apoptosis-inducer [8], [9], [10], [11]. More information on the extra-glycolytic roles of GAPDH can be found in the review by Nichollis et al. [12].

Despite decades of work on GAPDH’s from prokaryotic and eukaryotic sources, no work has been conducted on the GAPDH from M. tuberculosis. It was discovered early on in our work that this enzyme had significant solubility issues. This obstacle was overcome by co-transforming the Mtb-GAPDH plasmid along with a plasmid expressing the chaperones GroEL/GroES [13]. These chaperones are believed to create an environment suitable for proper folding yielding soluble and active Mtb-GAPDH [13]. In this study, we report the first successful purification and mechanistic evaluation of Mtb-GAPDH using steady-state kinetics, pH-rate profiles, isotope effects and mutagenesis to elucidate both the kinetic and chemical mechanism of Mtb-GAPDH.

Section snippets

Materials

All chemicals were purchased from Sigma–Aldrich unless otherwise noted. The Mtb-GAPDH gene was cloned into the Novagen pET-28a(+) vector. The GroEL/GroES plasmid was a gift from the Shrader Lab [13]. Primers were purchased from Invitrogen. All cloning enzymes and T7 competent Escherichia coli were purchased from New England Biolabs. Complete EDTA-free protease inhibitor cocktail and DNase were purchased from Roche. 99.9% deuterated water was purchased from Cambridge Isotope Laboratories.

Cloning, expression and purification of Mtb-GAPDH

The M.

Cloning, expression and purification

Mtb-GAPDH was PCR amplified then cloned into the pET28a expression vector encoding a N-terminal His6-tagged Mtb-GAPDH. Sequencing confirmed that no mutations were introduced during the cloning process. Initial expression studies revealed that Mtb-GAPDH was insoluble under normal conditions. Co-transformation of the Mtb-GAPDH plasmid along with a GroEL/GroES plasmid yielded soluble and active protein. After purification and dialysis, the final protein preparation was >95% pure as determined by

Chemical mechanism

The chemical mechanism for Mtb-GAPDH shown in Scheme 3 is supported by our determination of the kinetic mechanism, mutagenesis and isotope effect studies. The “free enzyme” is actually present with bound NAD+, and the two important catalytic residues, Cys158 and His185, are present as a thiolate-imidazolium ion pair. Binding of glyceraldehyde-3-phosphate yields an initial Michaelis complex, and the Cys158 thiolate nucleophilicially attacks the aldehyde to generate the neutral thiohemiacetal

Acknowledgments

We thank Drs. Subray Hegde and Hector Serrano for the assistance in the purification of Mtb-GAPDH. This work was supported by the National Institutes of Health Grant A1060899 to J.S.B.

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