Slow-Binding Inhibition of Escherichia coli Cystathionine β-Lyase by l-Aminoethoxyvinylglycine:  A Kinetic and X-ray Study

Abstract

The pyridoxal 5'-phosphate (PLP)-dependent cystathionine beta-lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kinetic methods and X-ray crystallography. Upon AVG treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102-105] was observed due to the generation of a long-lived, slowly dissociating enzyme-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (CBL + AVG --> CBLAVG, Ki = 1.1 +/- 0.3 microM) with an association rate constant (k1) of 336 +/- 40 M-1 s-1. This value is several orders of magnitude lower than typical bimolecular rate constants of ES formation, suggesting that additional steps occur before formation of the first detectable CBLAVG complex. Loss of activity is paralleled by the conversion of the pyridoxaldimine 426 nm chromophore to a 341 nm-absorbing species. On the basis of the recently solved structure of native CBL [Clausen, T., et al. (1996) J. Mol. Biol. 262, 202-224], it was possible to elucidate the X-ray structure of the CBLAVG complex and to refine it to an R-factor of 16.4% at 2.2 A resolution. The refined structure reveals the geometry of the bound inhibitor and its interactions with residues in the active site of CBL. Both the X-ray structure and the absorbance spectrum of the CBLAVG complex are compatible with a ketimine as the reaction product. Thus, the inhibitor seems to bind in a similar way to CBL as the substrate, but after alpha-proton abstraction, the reaction proceeds in a CBL nontypical manner, i.e. protonation of PLP-C4', resulting in the "dead-end" ketimine PLP derivative. The CBLAVG structure furthermore suggests a binding mode for rhizobitoxine and explains the failure of MVG to inhibit CBL.

Exploration of the active site of Escherichia coli cystathionine γ‐synthase

“…Substitution of the side chain of D116, located 8.3 Å from the distal amino group of AVG, in the structure of the eCBL complex with this inhibitor, does not modify the kinetic parameters of eCBL. 7,8 Similarly, the side-chain of R167 of the plant nCGS enzyme, which corresponds to eCGS-R106 and eCBL-D116, is oriented away from the phosphonate moiety of the inhibitor in the nCGS-APPA complex (Fig. 2), suggesting that although this residue likely does not bind the distal phosphate group of the L-OPHS substrate, it may interact with the a-carboxylate of L-Cys.…”

“…The conformation and orientation of substrate(s) within the active site as well as the degree of freedom of rotation about the Ca-Cb bond, of the substrate covalently bound to the cofactor, have been proposed to be determinants of reaction specificity among the enzymes of the c-subfamily of fold-type I of PLP-dependent enzymes. 7,8 However, these factors are subtle and not evident from comparison of the available crystal structures of the unliganded or inhibitor-bound complexes of these enzymes, from a mixture of bacterial and plant species, as a conserved residue may play different roles dependent on the context of the specific active site. Additionally, interconversion of the two residues most distinct between the active sites of the a,c-elimination catalyzing eCGS (D45 and E325) and yCGL (E48 and E333) and eCBL (F55 and Y338), which catalyzes an a,b-elimination reaction, does not result in a corresponding increase in a,c or a,b-elimination activity.…”

“…A similar 80-fold increase in K l-Cth m has been reported for the corresponding R372K variant of eCBL, which also interacts with the a-carboxylate moiety of the inhibitor in the eCBL-AVG complex. 7,8 The !220-fold decrease in the k cat of the a,c-replacement activity of eCGS-R361K is in keeping with the 55-fold decrease in this parameter reported for the corresponding AATase-R386K but contrasts with the 4-fold decrease in the k cat of CBL-R372K. 7,11,12 The observed reductions in the activity of the eCGS and eAAT variants reflects differences in the binding orientation and conformation of the substrates, such that they are not optimally positioned for catalysis.…”

Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine

“…1. Clausen et al (16) and Krupka et al (17) also find covalent AVG⅐PLP adducts in Escherichia coli cystathionine-␤-lyase (CBL) and in Treponema denticola cystalysin (PDB codes 1CL2 and 1C7O), respectively. Biochemical Evidence for a Ketimine AVG⅐PLP AdductIncubation of ACC synthase with AVG results in the rapid disappearance of the PLP internal aldimine absorption band at 421 nm with concomitant appearance of an enzyme species that absorbs at 341 nm (Fig.…”

Structure and Function of Threonine Synthase from Yeast

“…One monomer of the refined yTS x-ray structure was chosen as the starting point for model building and energy minimization of the modeled complex. OPHS and subsequently its external aldimine with PLP were generated and positioned into the active site based on the binding of aminoethoxy vinylglycine in cystathionine ␤-lyase (35). The Consistent Valence Forcefield of Insight II program was used for energy minimization with Discover3, which was run over 1000 minimization steps until convergence (0.1 kcal/mol tolerance).…”