L-asparaginase of Thermus Thermophilus: Purification, Properties and Identification of Essential Amino Acids for Its Catalytic Activity

Overview

Journal Mol Cell Biochem

Publisher Springer

Specialty Biochemistry

Date 2001 Feb 24

PMID 11216870

Citations 17

Authors

A A Pritsa

D A Kyriakidis

Affiliations

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Abstract

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77 degrees C. Chemical modification experiments implied the existence ofhistidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.

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References

Zhang S, McCarter J, Okamura-Oho Y, Yaghi F, Hinek A, Withers S . Kinetic mechanism and characterization of human beta-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells. Biochem J. 1994; 304 ( Pt 1):281-8. PMC: 1137483. DOI: 10.1042/bj3040281. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Distasio J, Niederman R, Kafkewitz D, Goodman D . Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes. J Biol Chem. 1976; 251(22):6929-33. View

Claassen E, van Rooijen N . A comparative study on the effectiveness of various procedures for attachment of two proteins (L-asparaginase and horse radish peroxidase) to the surface of liposomes. Prep Biochem. 1983; 13(2):167-74. DOI: 10.1080/00327488308068746. View

Fishman Y, CITRI N . L-asparaginase entrapped in liposomes: preparation and properties. FEBS Lett. 1975; 60(1):17-20. DOI: 10.1016/0014-5793(75)80408-x. View

Harms E, Wehner A, Aung H, Rohm K . A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis. FEBS Lett. 1991; 285(1):55-8. DOI: 10.1016/0014-5793(91)80723-g. View

WRISTON Jr J, Yellin T . L-asparaginase: a review. Adv Enzymol Relat Areas Mol Biol. 1973; 39:185-248. DOI: 10.1002/9780470122846.ch3. View

Melchior Jr W, FAHRNEY D . Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with alpha-chymotrypsin, pepsin, and pancreatic ribonuclease at pH 4. Biochemistry. 1970; 9(2):251-8. DOI: 10.1021/bi00804a010. View

Belleau B, Malek G . A new convenient reagent for peptide syntheses. J Am Chem Soc. 1968; 90(6):1651-2. DOI: 10.1021/ja01008a045. View

10.

Kafkewitz D, Bendich A . The inhibition of lymphocyte mitogenesis by asparaginase: a still unexplained phenomenon. Experientia. 1984; 40(11):1173-7. DOI: 10.1007/BF01946642. View

11.

Goldstein I, HOLLERMAN C, MERRICK J . PROTEIN-CARBOHYDRATE INTERACTION. I. THE INTERACTION OF POLYSACCHARIDES WITH CONCANAVALIN A. Biochim Biophys Acta. 1965; 97:68-76. DOI: 10.1016/0304-4165(65)90270-9. View

12.

Pritsa A, Choli-Papadopoulou T, Kyriakidis D . Studies on the primary structure of L-asparaginase from Thermus thermophilus. J Protein Chem. 1998; 17(6):548-9. View

13.

Kyriakidis D, Tsirka S, Tsavdaridis I, Iliadis S, Kortsaris A . Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines. Mol Cell Biochem. 1990; 96(2):137-42. DOI: 10.1007/BF00420905. View

14.

Adamson R, Fabro S . Antitumor activity and other biologic properties of L-asparaginase (NSC-109229)-a review. Cancer Chemother Rep. 1968; 52(6):617-26. View

15.

Bagert U, Rohm K . On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies. Biochim Biophys Acta. 1989; 999(1):36-41. DOI: 10.1016/0167-4838(89)90026-5. View

16.

Tsirka S, Kyriakidis D . In vitro alterations of L-asparaginase activity of Tetrahymena pyriformis by lipids. Mol Cell Biochem. 1988; 83(2):147-55. DOI: 10.1007/BF00226142. View

17.

Manna S, Sinha A, Sadhukhan R, Chakrabarty S . Purification, characterization and antitumor activity of L-asparaginase isolated from Pseudomonas stutzeri MB-405. Curr Microbiol. 1995; 30(5):291-8. DOI: 10.1007/BF00295504. View

18.

Distasio J, Salazar A, Nadji M, Durden D . Glutaminase-free asparaginase from vibrio succinogenes: an antilymphoma enzyme lacking hepatotoxicity. Int J Cancer. 1982; 30(3):343-7. DOI: 10.1002/ijc.2910300314. View

19.

Schwartz R . Immunosuppression by L-asparaginase. Nature. 1969; 224(5216):275-6. DOI: 10.1038/224275a0. View

20.

Tsirka S, Kyriakidis D . A model for the regulation of the activity of L-asparaginase/ kinase enzyme of Tetrahymena pyriformis. Biochem Int. 1989; 19(1):9-17. View