Kaartinen, Vesa and Williams, Julian C. and Tomich, John and Yates, John R., III and Hood, Leroy E. and Mononen, Ilkka (1991) Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline. Journal of Biological Chemistry, 266 (9). pp. 5860-5869. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:KAAjbc91
See Usage Policy.
Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:KAAjbc91
The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 184.108.40.206) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l^-1 x min^-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19- kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L- norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10^(3) M^-1 min^-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L- norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.
|Additional Information:||Copyright © 1991 by the American Society for Biochemistry and Molecular Biology. (Received for publication, June 5, 1990) We are grateful to Prof. I. Penttila, Department of Clinical Chemistry, Kuopio University Central Hospital, SF-70210 Kuopio, Finland, for placing the facilities of the Department at our disposal for purification and characterization of glycoasparaginase. Dr. J.S. Holcenberg, Division of Hematology and Oncology, Children's Hospital of Los Angeles is acknowledged for his advice and discussions. This work was supported by the Finnish Academy of Sciences and by National Science Foundation, Science and Technology Center Cooperative Grant 8809710 (to J.R.Y. and to L.E.H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Archive Administrator|
|Deposited On:||11 Sep 2007|
|Last Modified:||26 Dec 2012 09:41|
Repository Staff Only: item control page