STRUCTURE-ACTIVITY-RELATIONSHIPS AND THERMAL-STABILITY OF HUMAN GLUTATHIONE TRANSFERASE P1-1 GOVERNED BY THE H-SITE RESIDUE-105

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino a cid residue 105, positioned in the substrate binding II-site. To eluci date the role of this residue an extensive characterization of GSTP1-1 /Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/T rp105, were constructed and included in the study. Steady-state kineti c parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters i ndicates that the effect of the substitution of the amino acid in posi tion 105 is highly dependent on substrate us ed. When 1-chloro-2,4-din itrobenzene was used as substrate a change in the side-chain of residu e 105 seemed primarily to cause changes in the K-M,value, while the k( cat) value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both k (cat) and K-M values were altered by the substitution of amino acid 10 5. The constant for formation of the sigma-complex between 1,3,5-trini trobenzene and glutathione was shown to be dependent upon the volume o f the amino acid in position 105. The nature of the amino acid in posi tion 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/ Ile105 variant was approxima tely two to three times more stable than the Val105 variant as judged by their half-lives. The presence of,glutathione in the incubation buf fer afforded a threefold increase in the half-lives of the enzymes. Th us, tl-le thermal stability of the enzyme and depending on substrate, both K-M values and turnover numbers are influenced by substitutions i n position 105 of GSTP1-1. (C) 1998 Academic Press Limited.