Irreversible inactivation of snake venom l-amino acid oxidase by covalent modification during catalysis of l-propargylglycine

Abstract

Snake venom l-amino acid oxidase (SV-LAAO, a flavor-enzyme) has attracted considerable attention due to its multifunctional nature, which is manifest in diverse clinical and biological effects such as inhibition of platelet aggregation, induction of cell apoptosis and cytotoxicity against various cells. The majority of these effects are mediated by H2O2 generated during the catalytic conversion of l-amino acids. The substrate analog l-propargylglycine (LPG) irreversibly inhibited the enzyme from Crotalus adamanteus and Crotalus atrox in a dose- and time-dependent manner. Inactivation was irreversible which was significantly protected by the substrate l-phenylalanine. A Kitz-Wilson replot of the inhibition kinetics suggested formation of reversible enzyme-LPG complex, which occurred prior to modification and inactivation of the enzyme. UV-visible and fluorescence spectra of the enzyme and the cofactor strongly suggested formation of covalent adduct between LPG and an active site residue of the enzyme. A molecular modeling study revealed that the FAD-binding, substrate-binding and the helical domains are conserved in SV-LAAOs and both His223 and Arg322 are the important active site residues that are likely to get modified by LPG. Chymotrypsin digest of the LPG inactivated enzyme followed by RP-HPLC and MALDI mass analysis identified His223 as the site of modification. The findings reported here contribute towards complete inactivation of SV-LAAO as a part of snake envenomation management.

Keywords: CHD, 1,2-cyclohexanedione; Crotalus adamanteus venom; Crotalus atrox venom; DEPC, diethylpyrocarbonate; FAD, flavin adenine dinucleotide; Gdn-HCl, guanidine hydrochloride; Irreversible inactivation; LAAO, l-amino acid oxidase (EC. 1.4.3.2); LPG, l-propargylglycine; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; Mechanism-based inhibitor; TNBS, trinitrobenzene sulfonic acid.; l-Amino acid oxidase; l-Phe, l-phenylalaine; l-Propargylglycine.

Fig. 1

Catalytic oxidation of l-amino acid oxidase. The first half reaction involves the conversion of FAD to FADH₂ with concomitant oxidation of the amino acid to an imino acid. Another oxidative half reaction completes the catalytic cycle by reoxidizing the FADH₂ with oxygen, producing hydrogen peroxide. The unstable imino acid is then hydrolyzed by the enzyme bound water to form the final product keto acid.

Fig. 2

(A) Time course of oxidation of (a) 62.5, (b) 125, (c) 250, (d) 375 and (e) 500 μM of LPG by 81.22 nM of LAAO. Experimental conditions have been mentioned in the text. (B) Linear dependence of the initial rate and (C) product formation at 180 min, when the reactions was assumed to be complete with the concentration of LPG. R² (regression coefficient) values are 0.992 and 0.996, respectively.

Fig. 3

Dependence of irreversible inactivation of LAAO on the concentration of LPG. (a) Residual activity of LAAO after incubation with 0–25 mM of LPG at 4 °C for 24 h. (b) Activity of the enzyme preparations of set (a) after passing through Sephadex G-50 ‘Spin column’ to remove unreacted LPG and its catalytic products. (c) Activity of the enzyme preparation of set (b) after incubation at 4 °C for 24 h allowing time dependent dissociation of enzyme–inhibitor adduct, if any. Activity of the enzyme before passing through ‘Spin-column’ is taken as 100%.

Fig. 4

Presentation of the mechanism of inactivation of LAAO, presented as E, by the suicidal substrate LPG. The ratio k_cat/k_inact is define as the partition ratio and it denotes the inhibitor potency of the inhibitor, as it express the extent of bifurcation of a reaction towards product formation and inactivation.

Fig. 5

Protection of LAAO activity with concentration of l-Phe in presence of 25 mM of LPG. Activity of the enzyme incubated with 0.38 mM l-Phe is considered as 100%.

Fig. 6

(A) Time dependent inactivation of LAAO with variable concentration of LPG at 30 °C. (B) Kitz–Wilson replot from the adjacent kinetic data correlating the inverse of k_obs with the inverse of LPG concentration.

Fig. 7

(A) UV–visible spectra of FAD (13 μM) (—), LAAO dialyzed against buffer (- - -), LAAO inactivated by LPG before (·····) and after dialysis (-·-·-). LAAO concentration was 0.5 U/ml. (B) UV–visible spectra of apo-LAAO (—) and LPG inactivated apo-LAAO (- - -). The later shows a distinct peak at 320 nm marked by arrow. (C) Emission and (D) excitation spectra of FAD from control LAAO (—) and LPG inactivated LAAO (- - -). These spectra are identical with that of reference FAD that had not been included for clarity.

Fig. 8

(A) Sequence alignment of the catalytic region of LAAOs from different venom source, indicating conserved positions of histidine and arginine residues (marked by arrows). The FAD and substrate binding residues are highlighted in light black and blue respectively while yellow shade represents both substrate and FAD binding residues. (B) Homology model of C. adamanteus LAAO obtained from CPH model 3.0 online server. The catalytic site residues have been shown and they are deeply buried within the enzyme. (C) Molecular surface representation of LAAO is used to help visualization of the catalytic funnel where LPG being docked at the position of normal substrate, maintaining a distance of 3.5 Å between α-C atom of LPG (cyan) and flavine (red). (D) Stereo view of the catalytic site of LAAO showing the environment of LPG. The substrate LPG is shown in cyan and the FAD in yellow. His223 and Arg322 that are susceptible to modification are present in the side-chain binding sub-pocket and nearest to the accetylenic moiety of LPG. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

Residual activity of LAAO (%) after modification with DEPC, CHD and TNBS in presence and absence of l-Phe or LPG.

Fig. 10

Proposed mechanism to explain the catalytic and suicide inactivation pathway of l-amino acid oxidase by LPG. LPG (1) is oxidised by LAAO to the acetylenic imine intermediate (2). The intermediate (2) can rearranged to the allenic imine intermediate (3), which either cyclises through intramolecular nucleophilic attack to give the product 3-amino-5-methylene-5H-furan-2-one (4) or undergo nucleophilic attack by a nucleophile present at the active site of the enzyme to form the inactive enzyme–inhibitor adduct (5).

Fig. 11

(A) RP-HPLC chromatogram of chymotryptic digest of LAAO inactivated by LPG monitored at 220 nm. Inset. The same chromatogram of control LAAO (upper) and that of LPG inactivated LAAO (lower) respectively monitored at 320 nm. The peak of R_t 29.4 min marked by arrow corresponds to the LPG modified peptide. (B) MALDI mass spectrum shows m/z of 884.89 Da, which correspond to the modified peptide KH*DDIF where * denotes LPG adduct. The MSMS spectrum of this marked peptide is shown in Supplementary Fig. S4.