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

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Redox Biology Center and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606
†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.
* Corresponding author. Ruma Banerjee, 3320 B, MSRB III, 1150 W. Medical Center Dr., University of Michigan, Ann Arbor, MI 48109-0606. Tel: (734)-615-5238 . Fax: (734)-763-7799. E-mail:[email protected]
‡University of Nebraska.
§University of Michigan.
Cite this: Biochemistry 2008, 47, 23, 6226–6232
Publication Date (Web):May 14, 2008
https://doi.org/10.1021/bi800351a
Copyright © 2008 American Chemical Society

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    Abstract

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    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 Vmax compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in Vmax. The KMs 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.

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