Kinetic Properties of Polymorphic Variants and Pathogenic Mutants in Human Cystathionine γ-Lyase

Abstract

Human cystathionine-γ-lyase (CGL) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme, which functions in the transsulfuration pathway that converts homocysteine to cysteine. In addition, CGL is one of two major enzymes that can catalyze the formation of hydrogen sulfide, an important gaseous signaling molecule. Recently, several mutations in CGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Moreover, a common single nucleotide polymorphism in CGL, c.1364G>T that converts serine at position 403 to isoleucine, has been linked to elevated plasma homocysteine levels. In this study, we have characterized the pathogenic T67I and Q240E missense mutations and the polymorphic variants at amino acid residues 403 using kinetic and spectrophotometric methods. We report that the polymorphism does not influence the cofactor content of the enzyme or its steady-state kinetic properties. In contrast, the T67I mutant exhibits a 3.5-fold decrease in V max compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in V max . The K M s for cystathionine for both pathogenic mutants are comparable to that of wild type CGL. The PLP content of the T67I and Q240E mutants were about 4-fold and 80-fold lower than that of wild-type enzyme, respectively. Preincubation of the T67I mutant with PLP restored activity to wild-type levels while the same treatment resulted in only partial restoration of activity of the Q240E mutant. These results reveal that both mutations weaken the affinity for PLP and suggest that cystathionuric patients with these mutations should be responsive to pyridoxine therapy.The mammalian transsulfuration pathway represents one of two major metabolic routes for the removal of homocysteine, which at elevated levels, is an independent risk factor for cardiovascular diseases and other complex disorders (1,2). Cystathionine γ-lyase (CGL 1 ), the second enzyme in the transsulfuration pathway, catalyzes the conversion of † This work was supported in part by a grant from the National Institutes of Health (HL58984 to R.B.) and by an American Heart Association Postdoctoral Fellowship to W.Z.

H2S Biogenesis by Human Cystathionine γ-Lyase Leads to the Novel Sulfur Metabolites Lanthionine and Homolanthionine and Is Responsive to the Grade of Hyperhomocysteinemia

“…The protein was purified as described previously (24) with the following modification. After the Superdex S-200 (Sigma) size exclusion column, the active fractions were pooled, concentrated, and dialyzed against 100 mM Hepes buffer, pH 7.4, before being stored at Ϫ80°C.…”

PLP-Dependent Enzymes: a Potent Therapeutic Approach for Cancer and Cardiovascular Diseases

“…By cystathioninesynthase, homocysteine condensates with serine moiety forming cystathionine, that subsequently oxidized to cysteine, -ketobutyrate and ammonia by cystathionine -lyase (Zou and Baerjee, 2005). Biochemically, transsulfuration pathway contributes in maintaining the cellular balance of cysteine-homocysteine pool that participates in about 50% of total antioxidant formation (Zhu et al, 2008). Practically, inactivation of cystathionine -synthase causes hyperaccumulation of homocysteine that is a visual risk of cardiovascular diseases, damage to vascular endothelia (De Bree et al, 2002, Wald et al, 2002) and Alzheimer's disease (Morris, 2003).…”

“…P. ovalis L-methioninase lacks the ability to restore its original activity by dialysis against pyridoxal phosphate (Johnston et al, 1979), while that of Trichomonas vaginalis enzyme restore more than 90 % of its activity by 0.1mM PLP (Lockwood and Coombs, 1991). However, A. flavipes L-methioninase has the ability to reconstitute its fully structural catalytic state upon addition of pyridoxal phosphate (0.2mM), similarly to cystathioninelyase (Zhu et al, 2008). …”

“…Dietary therapy relying of deprivation of methionine and supplementation of cysteine display a reliable positive result in delaying the clinical manifestations (Pullon, 1980). However, supplementation of cystathioninesynthase was the potent relevant therapy for dramatically conversion of homocysteine to cystathionine (Zhu et al, 2008). Since cystathionine -synthase is a PLP-dependent enzyme and the rapid dissociation of PLP as coenzyme from the apoenzyme is a common structural and catalytic criteria (El-Sayed, 2010, 2011, external supplementation of pyridoxal phosphate, vitamin B6 gave a plausible results for decreasing the amount of homocysteine, assuming the reassociation of apo-cystathionine -synthase forming the holo-enzyme (Barber and Spaeth, 1967).…”

“…Since cystathionine -synthase is a PLP-dependent enzyme and the rapid dissociation of PLP as coenzyme from the apoenzyme is a common structural and catalytic criteria (El-Sayed, 2010, 2011, external supplementation of pyridoxal phosphate, vitamin B6 gave a plausible results for decreasing the amount of homocysteine, assuming the reassociation of apo-cystathionine -synthase forming the holo-enzyme (Barber and Spaeth, 1967). Moreover, the deficiency of cystathionine -lyase results in accumulation of cystathionine (Cystathioninuria) that usually accompanied with diabetes, Down's syndrome, neuroblastoma (Wang and Hegele, 2003) as reviewed by Zhu et al (2008).…”