Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 15;328(Pt 1):231–235. doi: 10.1042/bj3280231

Mutagenesis of residue 157 in the active site of human glyoxalase I.

M Ridderström 1, A D Cameron 1, T A Jones 1, B Mannervik 1
PMCID: PMC1218911  PMID: 9359858

Abstract

Met-157 in the active site of human glyoxalase I was changed by site-directed mutagenesis into alanine, glutamine or histidine in order to evaluate its possible role in catalysis. The glyoxalase I mutants were expressed in Escherichia coli and purified on an S-hexylglutathione affinity gel. The physicochemical properties of the mutant proteins were similar to those of the wild-type enzyme. The glutamine mutant exhibited the same high specific activity as wild-type glyoxalase I, whereas the alanine and histidine mutants had approx. 20% of wild-type activity. The kcat/Km values of the mutant glyoxalase I determined with the hemithioacetal adduct of glutathione and methylglyoxal were reduced to between 10 and 40% of the wild-type value. This reduction was due to lower kcat values for the alanine and histidine mutants and a twofold increase in the Km value for the glutamine mutant. With the hemithioacetal of glutathione and phenylglyoxal, the kinetic parameters of the mutants were also of the same magnitude as those of wild-type glyoxalase I. Studies with the competitive inhibitors S-hexyl- and S-benzyl-glutathione revealed that the affinity was reduced to 7-11% of the wild-type affinity for the glutamine and alanine mutants and to 30-40% for the histidine mutant, as measured by a comparison of Ki values. The results show that Met-157 has no direct role in catalysis, but is rather involved in forming the substrate-binding site of human glyoxalase I. The high activity of the glutamine mutant suggests that a structurally equivalent glutamine residue in the N-terminal half of Saccharomyces cerevisiae glyoxalase I may be part of a catalytically competent active site.

Full Text

The Full Text of this article is available as a PDF (249.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aronsson A. C., Mannervik B. Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography. Biochem J. 1977 Sep 1;165(3):503–509. doi: 10.1042/bj1650503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aronsson A. C., Marmstål E., Mannervik B. Glyoxalase I, a zinc metalloenzyme of mammals and yeast. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1235–1240. doi: 10.1016/0006-291x(78)91268-8. [DOI] [PubMed] [Google Scholar]
  3. Aronsson A. C., Tibbelin G., Mannervik B. Purification of glyoxalase I from human erythrocytes by the use of affinity chromatography and separation of the three isoenzymes. Anal Biochem. 1979 Jan 15;92(2):390–393. doi: 10.1016/0003-2697(79)90676-6. [DOI] [PubMed] [Google Scholar]
  4. Björnestedt R., Widersten M., Board P. G., Mannervik B. Design of two chimaeric human-rat class alpha glutathione transferases for probing the contribution of C-terminal segments of protein structure to the catalytic properties. Biochem J. 1992 Mar 1;282(Pt 2):505–510. doi: 10.1042/bj2820505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cameron A. D., Olin B., Ridderström M., Mannervik B., Jones T. A. Crystal structure of human glyoxalase I--evidence for gene duplication and 3D domain swapping. EMBO J. 1997 Jun 16;16(12):3386–3395. doi: 10.1093/emboj/16.12.3386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casazza J. P., Felver M. E., Veech R. L. The metabolism of acetone in rat. J Biol Chem. 1984 Jan 10;259(1):231–236. [PubMed] [Google Scholar]
  7. Espartero J., Sánchez-Aguayo I., Pardo J. M. Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress. Plant Mol Biol. 1995 Dec;29(6):1223–1233. doi: 10.1007/BF00020464. [DOI] [PubMed] [Google Scholar]
  8. Inoue Y., Kimura A. Identification of the structural gene for glyoxalase I from Saccharomyces cerevisiae. J Biol Chem. 1996 Oct 18;271(42):25958–25965. [PubMed] [Google Scholar]
  9. Jerzykowski T., Winter R., Matuszewski W., Piskorska D. A re-evaluation of studies on the distribution of glyoxalases in animal and tumour tissues. Int J Biochem. 1978;9(11):853–860. doi: 10.1016/0020-711x(78)90036-8. [DOI] [PubMed] [Google Scholar]
  10. Kellum M. W., Oray B., Norton S. J. A convenient quantitative synthesis of methylglyoxal for glyoxalase I assays. Anal Biochem. 1978 Apr;85(2):586–590. doi: 10.1016/0003-2697(78)90258-0. [DOI] [PubMed] [Google Scholar]
  11. Kim N. S., Sekine S., Kiuchi N., Kato S. cDNA cloning and characterization of human glyoxalase I isoforms from HT-1080 cells. J Biochem. 1995 Feb;117(2):359–361. doi: 10.1093/jb/117.2.359. [DOI] [PubMed] [Google Scholar]
  12. Kim N. S., Umezawa Y., Ohmura S., Kato S. Human glyoxalase I. cDNA cloning, expression, and sequence similarity to glyoxalase I from Pseudomonas putida. J Biol Chem. 1993 May 25;268(15):11217–11221. [PubMed] [Google Scholar]
  13. Larsen K., Aronsson A. C., Marmstål E., Mannervik B. Immunological comparison of glyoxalase I from yeast and mammals and quantitative determination of the enzyme in human tissues by radioimmunoassay. Comp Biochem Physiol B. 1985;82(4):625–638. doi: 10.1016/0305-0491(85)90499-7. [DOI] [PubMed] [Google Scholar]
  14. Lo T. W., Westwood M. E., McLellan A. C., Selwood T., Thornalley P. J. Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin. J Biol Chem. 1994 Dec 23;269(51):32299–32305. [PubMed] [Google Scholar]
  15. Lu T., Creighton D. J., Antoine M., Fenselau C., Lovett P. S. The gene encoding glyoxalase I from Pseudomonas putida: cloning, overexpression, and sequence comparisons with human glyoxalase I. Gene. 1994 Dec 2;150(1):93–96. doi: 10.1016/0378-1119(94)90864-8. [DOI] [PubMed] [Google Scholar]
  16. NEMETH A. M., RUSSELL C. S., SHEMIN D. The succinate-glycine cycle. II. Metabolism of delta-aminolevulinic acid. J Biol Chem. 1957 Nov;229(1):415–422. [PubMed] [Google Scholar]
  17. RACKER E. The mechanism of action of glyoxalase. J Biol Chem. 1951 Jun;190(2):685–696. [PubMed] [Google Scholar]
  18. Ranganathan S., Walsh E. S., Godwin A. K., Tew K. D. Cloning and characterization of human colon glyoxalase-I. J Biol Chem. 1993 Mar 15;268(8):5661–5667. [PubMed] [Google Scholar]
  19. Richard J. P. Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance. Biochemistry. 1991 May 7;30(18):4581–4585. doi: 10.1021/bi00232a031. [DOI] [PubMed] [Google Scholar]
  20. Ridderström M., Mannervik B. Optimized heterologous expression of the human zinc enzyme glyoxalase I. Biochem J. 1996 Mar 1;314(Pt 2):463–467. doi: 10.1042/bj3140463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ridderström M., Mannervik B. The primary structure of monomeric yeast glyoxalase I indicates a gene duplication resulting in two similar segments homologous with the subunit of dimeric human glyoxalase I. Biochem J. 1996 Jun 15;316(Pt 3):1005–1006. doi: 10.1042/bj3161005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sellin S., Eriksson L. E., Mannervik B. Fluorescence and nuclear relaxation enhancement studies of the binding of glutathione derivatives to manganese-reconstituted glyoxalase I from human erythrocytes. A model for the catalytic mechanism of the enzyme involving a hydrated metal ion. Biochemistry. 1982 Sep 28;21(20):4850–4857. doi: 10.1021/bi00263a004. [DOI] [PubMed] [Google Scholar]
  24. Thornalley P. J., McLellan A. C., Lo T. W., Benn J., Sönksen P. H. Negative association between erythrocyte reduced glutathione concentration and diabetic complications. Clin Sci (Lond) 1996 Nov;91(5):575–582. doi: 10.1042/cs0910575. [DOI] [PubMed] [Google Scholar]
  25. Thornalley P. J. The glyoxalase system in health and disease. Mol Aspects Med. 1993;14(4):287–371. doi: 10.1016/0098-2997(93)90002-u. [DOI] [PubMed] [Google Scholar]
  26. Vaca C. E., Fang J. L., Conradi M., Hou S. M. Development of a 32P-postlabelling method for the analysis of 2'-deoxyguanosine-3'-monophosphate and DNA adducts of methylglyoxal. Carcinogenesis. 1994 Sep;15(9):1887–1894. doi: 10.1093/carcin/15.9.1887. [DOI] [PubMed] [Google Scholar]
  27. Vander Jagt D. L., Han L. P., Lehman C. H. Kinetic evaluation of substrate specificity in the glyoxalase-I-catalyzed disproportionation of -ketoaldehydes. Biochemistry. 1972 Sep 26;11(20):3735–3740. doi: 10.1021/bi00770a011. [DOI] [PubMed] [Google Scholar]
  28. Vince R., Daluge S., Wadd W. B. Studies on the inhibition of glyoxalase I by S-substituted glutathiones. J Med Chem. 1971 May;14(5):402–404. doi: 10.1021/jm00287a006. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES