Kinetics of the exchange reaction catalyzed by 2-amino-3- ketobutyrate CoA ligase

Abstract

2-Amino-3-ketobutyrate CoA ligase (KBL) of Escherichia coli is a member of the α-oxoamine synthase family; it catalyzes the condensation reaction between glycine and acetyl CoA to yield 2-amino-3-ketobutyrate. We have previously shown that KBL catalyzes the exchange of pro-R hydrogen of glycine with protons in the medium; however, the kinetics of this reaction has never been determined. In this study, we calculated the kinetic parameters of this exchange reaction by using different concentrations of [2RS- ³H₂: 2-¹⁴C] glycine. The rate of the exchange reaction was determined by measuring the ³H/¹⁴C ratio in recovered [2S- ³H: 2-¹⁴C]glycine. The Lineweaver-Burk plot showed that K_m and k_cat of this reaction were 3.8 ×10^-3 M and 0.22 S^-1, respectively. On the other hand, K_m and k_cat values of the overall KBL-mediated catalysis were correspondingly 1.23 × 10^-2 M and 1.19 S^-1. Thus, the rate of the exchange reaction was almost five times lower than that of overall KBL catalysis.

Keywords

enzyme / 2-amino-3-ketobutyrate CoA ligase / kinetics / exchange reaction

Introduction

The pyridoxal phosphate (PLP)-dependent enzyme 2-amino-3-ketobutyrate CoA ligase (KBL) catalyzes the condensation reaction between glycine and acetyl CoA to yield 2-amino-3-ketobutyrate ( Mukherjee and Dekker, 1987; Marcus and Dekker, 1993; Schmidt et al., 2001). This enzyme has been characterized in different prokaryotes and eukaryotes, such as Escherichia coli and humans, and its role in threonine degradation/synthesis has been well established both in vivo and in vitro ( Mukherjee and Dekker, 1987; Marcus and Dekker, 1993; Edgar and Polak, 2000).

KBL is one of the four members of the α-oxoamine synthase family. Other members of this family include 5-aminolevulinate synthase (ALAS) ( Gibson et al., 1958; Riddle et at., 1989), 8-amino-7-oxononanoate synthase (AONS) ( Alexeev et al., 1998; Kerbarh et al., 2006), and serine palmitoyltransferase (SPT) ( Hanada et al.,1998; Gable et al., 2000; Hanada et al., 2000). These enzymes play an important role in biosynthesis of sphingolipids, tetrapyrroles, and biotin. All four enzymes catalyze a Claisen-type condensation reaction between different amino acids and acyl-CoA thioesters followed by a decarboxylation reaction ( Alexeev et al., 1998; Edgar and Polak, 2000) (supplement Scheme S1). However, KBL only catalyzes the condensation reaction and not the decarboxylation of 2-amino-3-ketobutyrate (Scheme 1), which is in contrast to the reactions catalyzed by the other three members of the α-oxoamine synthase family( Marcus and Dekker, 1993; Bashir et al., 2006). It has been shown that 2-amino-3-ketobutyrate is a very unstable intermediate that is either converted into threonine by L-threonine dehydrogenase (TDH) or decarboxylates (non-enzymatically) into aminoacetone ( Marcus and Dekker, 1993) (supplement Scheme S1).

Previously, our laboratory has shown that KBL from E. coli catalyzes exchange of pro-R of glycine with protons of the medium ( Bashir et al., 2006; Jamil et al., 2010), but the rate of this exchange reaction has never been determined (Scheme 1). Therefore, we sought to study kinetic parameters of this exchange reaction, which is one of the steps of the overall biochemical process catalyzed by KBL and TDH. Information about these parameters will contribute to a better understanding of the threonine degradation pathway.

Methods

Purification of KBL and TDH

Expression and purification of KBL were carried out according to methods outlined by Mukherjee and Dekker (1987) and Jamil et al.(2010). Recombinant KBL-pET and TDH-pET vectors were transformed and expressed in E. coli separately. Expression of both genes was induced by 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Translated proteins were soluble in water. The proteins were purified by using anion, hydrophobic, and gel filtration chromatography. The purified samples were analyzed by polyacrylamide gel electrophoresis (PAGE) followed by staining with Coomassie Brilliant Blue.

Molecular mass analysis of purified KBL

Molecular mass of purified KBL was obtained using a 6224 TOF LC/MS system (Agilent Technologies, USA) equipped with a dual electro-spray ionization source. Positive ions were produced by using a dual ESI-voltage of 3.5 kV at 325°C, gas flow 5 L/min, and nebulizer pressure of 30 psig. Data were collected at a rate of 1.03spectra/s. The flow injection analysis of the apoenzyme was performed by injecting 20 mL of the KBL solution (5 mg of protein/mL in 10 mM Tris-HCl, pH 8) at a flow-injection rate of 0.2 mL/min using a mixture of 0.1% formic acid (70%) and acetonitrile solution in 0.1% formic acid (30%). The multiply charged spectrum was extracted from TIC using Agilent Mass Hunter qualitative analysis software and processed for deconvolution.

Rate of exchange of pro-R hydrogen of [2RS-³H₂: 2-¹⁴C]glycine catalyzed by KBL

One milliliter of the reaction mixture that contained KBL (0.6 U) and [2RS-³H₂: 2-¹⁴C]glycine (different concentrations in different reactions) was incubated at 37°C in 50 mM Tris-HCl (pH 8.0). Aliquots (200 µL) were taken from the reaction mixture at 0, 15, 30, 60 and 120 min after the start of the reaction. The samples were placed in boiling water for 5 min to quench the reaction and converted to benzyloxycarbonyl glycine as described in Jamil et al. (2010). A portion of benzyloxycarbonyl glycine crystals was dissolved in the scintillation fluid for radioactivity measurements and changes in the ³H/¹⁴C ratio were used to calculate the rate of the exchange reaction.

The rate of the coupled reaction of KBL and TDH

The overall catalytic activity of the recombinant KBL was assayed as described by Marcus and Dekker (1993). The reaction mixture contained 50 mM Tris-HCl (pH 8.0), 200 mM glycine, 1 mM acetyl CoA, 0.5 mM NADH, KBL (0.6U), and TDH (1U) in a final volume of 1 mL. This mixture was incubated at 37°C and changes in NADH absorbance were monitored at 340 nm (ϵ = 6.2 mM^–1·cm^–1) for 10 min. One unit was defined as the quantity of enzyme, which catalyzed the formation of 1 mmol NAD⁺ per min at 37°C. For kinetic measurements, different glycine concentrations were used in separate reactions to calculate K_m and V_max values.

Results and discussion

Induction with 0.2 mM IPTG induced KBL expression in E. coli but the enzyme was inactive. After sonication, the cell extract was incubated with 0.1 mM PLP in 50 mM Tris HCl buffer (pH 8.0). After activation, the enzyme was purified by precipitation with 45% ammonium sulfate followed by anion and hydrophobic column chromatography. The purified enzyme produced a single 43 kDa band on the 12% SDS PAGE gel (Fig. 1). Analysis of purified KBL by MALDI-TOF indicated that its molecular mass was 43,117.15 ͟±1 Da (Fig. 2). This value perfectly matched previous KBL amino acid sequence data reported by Mukherjee and Dekker (1987).

The rate of the exchange reaction

The incubation of [2RS-³H₂: 2-¹⁴C]glycine with KBL resulted in a gradual decrease of the ³H/¹⁴C ratio of recovered glycine. This decrease may be attributed to the exchange reaction catalyzed by KBL, in which pro-R tritium is substituted by protons of the medium (Scheme 1). The reaction was carried out at different concentrations of [2RS-³H₂: 2-¹⁴C]glycine. We observed that the rate of the exchange reaction with 8 mM [2RS-³H₂: 2-¹⁴C]glycine was almost 20 times higher than that in presence of 2 mM glycine (Fig. 3). The method that was used for rate determination by an example in which 2 mM [2RS-³H₂: 2-¹⁴C]glycine (24137 ¹⁴C counts per min) was incubated with KBL, and an aliquot was removed after 10 min of incubation and converted to benzyoxyglycine for radioactivity loss measurement.

The ³H/¹⁴C ratio at start of reaction= 100%

After 15 min of incubation with KBL,

the ³H/¹⁴C ratio inrecovered benzyloxycarbonylglycine= 70%

The change in the ³H/¹⁴C ratio= 30%

The rate of the exchange reaction=

\frac{\mathrm{Decrease\; of}{}^{3}H{/}^{14}C(\%)\times \mathrm{\text{Conc. of the glycine used}}}{\mathrm{\text{Half of the}}{}^3\mathrm{H}{/}^{14}\mathrm{C}\mathrm{\text{ratio (50\%)}}\times \mathrm{\text{Time of reaction}}}

Therefore, the rate of the exchange reaction= (30 ×2) / (50 ×15) = 0.08 µmol/min

Similarly, the rates of the exchange reaction under different concentrations of [2RS-³H₂: 2-¹⁴C]glycine were obtained (Table 1) and the Lineweaver-Burk Plot showed that K_m (1/x-intercept: 1/0.26) and k_cat (1/y-intercept: 1/4.5) comprised 3.8 mM and 0.22 s^-1, respectively (Fig. 4).

The rate of the coupled reaction of KBL and TDH

The overall catalysis rate of KBL was determined by coupling it with TDH. In the coupled reaction, KBL catalyzed condensation of glycine and acetyl CoA to 2-amino-3-ketobutyrate that was reduced to threonine by TDH and NADH. By keeping concentrations of all substrates constant, it was observed that the rate of the coupled reaction accelerated with the increase in the concentration of glycine. The rate of the coupled reaction was almost 2.3 times higher in 25 mM glycine than that the rate obtained in presence of 5 mM glycine. By using coupled reaction rates calculated at different concentrations of glycine, the values of K_m (1/0.08) and k_cat (1/.84) were found to be12.3 mM and 1.19 s^-1, respectively.

This showed that the rate of exchange of glycine pro-R hydrogen comprised 18.5% of the overall KBL catalysis rate (Table 2). Such a slow rate could be attributed to the absence of acetyl CoA. In the case of ALAS, the rate of an identical process appeared to be very slow although it had not been determined quantitatively. For AONS, the rate of exchange was 10 times lower than the overall biosynthesis rate ( Ploux and Marquet, 1996). In comparison with these enzymes, the exchange of C-2 hydrogen of glycine catalyzed by KBL was apparently faster.

Abbreviations

KBL: 2-amino-3-ketobutyrate CoA ligase; ALAS: aminolevulinate synthase; AONS: 8-amino-7-oxononanoate synthase; SPT: serine palmitoyltransferase; TDH: L-threonine dehydrogenase, PAGE: polyacrylamide gel electrophoresis.

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