HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Metzler, D. E. Articles by Morino, Y. Search for Related Content PubMed PubMed Citation Articles by Metzler, D. E. Articles by Morino, Y. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 269, Issue 45, 28017-28026, Nov, 1994

NMR studies of 1H resonances in the 10-18-ppm range for cytosolic aspartate aminotransferase

DE Metzler, CM Metzler, ET Mollova, RD Scott, S Tanase, K Kogo, T Higaki and Y Morino
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.

Continuing a previous investigation (Kintanar, A., Metzler, C. M., Metzler, D. E., and Scott, R. D. (1991) J. Biol. Chem. 266, 17222- 17229), we have recorded 1H NMR spectra at 500 MHz in the 10-18-ppm range for the 93-kDa porcine cytosolic aspartate aminotransferase and for four specific mutant forms of the enzyme in which histidine 68 has been replaced by lysine or histidine 143, 189, or 193 has been replaced by glutamine. We have correlated resonances for apoenzyme, pyridoxamine and pyridoxal phosphate forms, and dicarboxylate complexes and have assigned imidazole NH resonances of active site histidines. The chemical shifts of several resonances undergo pH-dependent changes around the pKa of the Schiff base proton at the active site. Other resonances shift upon binding of dicarboxylates or other ligands. Phosphate or carboxylate ions, which can also occupy the site of the substrate's alpha-carboxylate, cause rapid exchange of the Schiff base proton. Although most resonances in the 10-18-ppm range disappear rapidly in D2O, a few are retained for months in the presence of the dicarboxylate inhibitor glutarate. We demonstrate that changes in chemical shifts and in exchange rates are sensitive indicators of electronic interactions of the enzyme with ligands and of conformational change. Nuclear Overhauser effects from NH protons have allowed us to identify resonances of CH protons of the imidazole rings of histidines 143, 189, and 193. Observed and predicted chemical shifts have been compared. We conclude that the net charge on this histidine cluster is zero but that some negative charge from the aspartate 222 carboxylate is donated inductively into the histidine 143 ring. Studies of the related enzyme from Escherichia coli are provided in an accompanying paper (Metzler, D. E., Metzler, C. M., Scott, R. D., Mollova, E. T., Kagamiyama, H., Yano, T., Kuramitsu, S., Hayashi, H., Hirotsu, K., and Miyahara, I. (1994) J. Biol. Chem. 269, 28027-28033). Our approach should be applicable to the study of active sites of a broad range of relatively large proteins.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. Kahyaoglu, K. Haghjoo, F. Guo, F. Jordan, C. Kettner, F. Felfoldi, and L. Polgar Low Barrier Hydrogen Bond Is Absent in the Catalytic Triads in the Ground State but Is Present in a Transition-state Complex in the Prolyl Oligopeptidase Family of Serine Proteases J. Biol. Chem., October 10, 1997; 272(41): 25547 - 25554. [Abstract] [Full Text] [PDF]

				S. A. Ahmed, P. McPhie, and E. W. Miles Mechanism of Activation of the Tryptophan Synthase alpha 2beta 2 Complex. SOLVENT EFFECTS OF THE CO-SUBSTRATE beta -MERCAPTOETHANOL J. Biol. Chem., November 15, 1996; 271(46): 29100 - 29106. [Abstract] [Full Text] [PDF]