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Characterization of site‐directed mutants of residues R58, R59, D116, W340 and R372 in the active site of E. coli cystathionine β‐lyase
Author(s) -
Lodha Pratik H.,
Jaworski Allison F.,
Aitken Susan M.
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.308
Subject(s) - chemistry , cystathionine beta synthase , active site , mutant , stereochemistry , hydrolysis , residue (chemistry) , biochemistry , enzyme , cysteine , gene
Cystathionine β‐lyase (CBL) catalyzes the hydrolysis of L ‐cystathionine ( L ‐Cth) to produce L ‐homocysteine, pyruvate, and ammonia. A series of active‐site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k cat / K m L ‐Cthfor the β‐elimination reaction range from a reduction of only 3‐fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L ‐Cth is: R372 >> R58 > W340 ≈ R59 > D116. Comparison of the kinetic parameters for L ‐Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the α‐carboxylate moiety. The increase in the p K a of the acidic limb and decrease in the p K a of the basic limb of the k cat / K m L ‐Cthversus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the p K a of an active‐site residue.