Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis

Abstract

Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A-A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively. The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database.

Binding and Kinetic Mechanisms of the Zeta Class Glutathione Transferase

“…The Zeta class GST is widely distributed in nature and contains a characteristic motif (SSCX(W/H)RVIAL) in the Nterminal region (11). Among these residues, the first three (Ser 14 -Ser 15 -Cys 16 ) line the active site pocket and have been investigated recently for their possible role in catalysis. In particular, the crystal structure of the human variant GSTZ1a-1a shows the sulfur atom of Cys 16 close to the sulfur atom of GSH (14).…”

“…Among these residues, the first three (Ser 14 -Ser 15 -Cys 16 ) line the active site pocket and have been investigated recently for their possible role in catalysis. In particular, the crystal structure of the human variant GSTZ1a-1a shows the sulfur atom of Cys 16 close to the sulfur atom of GSH (14). It has been shown that C16A mutant is active with different substrates but displays a higher K m value for GSH.…”

“…It has been shown that C16A mutant is active with different substrates but displays a higher K m value for GSH. Thus, Cys 16 seems to play a role in a proper binding of GSH rather than a direct role in catalysis (15). Four polymorphic variants of the human GSTZ1-1 have been detected in the Caucasian population resulting from mutations at positions 32, 42, and 82 (16,17).…”

“…Thus, Cys 16 seems to play a role in a proper binding of GSH rather than a direct role in catalysis (15). Four polymorphic variants of the human GSTZ1-1 have been detected in the Caucasian population resulting from mutations at positions 32, 42, and 82 (16,17). In the present study, binding and kinetic mechanisms of the most abundant variant, the human GSTZ1c-1c, have been carefully investigated using DCA and GSH as substrates.…”

“…In the present study, binding and kinetic mechanisms of the most abundant variant, the human GSTZ1c-1c, have been carefully investigated using DCA and GSH as substrates. Moreover, binding and kinetic properties of its C16A mutant have been determined by disclosing multiple roles for the Cys 16 residue.…”