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

This Article Full Text Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsui, I. Articles by Svensson, B. Search for Related Content PubMed PubMed Citation Articles by Matsui, I. Articles by Svensson, B. Social Bookmarking                      What's this?

Volume 272, Number 36, Issue of September 5, 1997 pp. 22456-22463
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Improved Activity and Modulated Action Pattern Obtained by Random Mutagenesis at the Fourth - Loop Involved in Substrate Binding to the Catalytic (/)₈-Barrel Domain of Barley -Amylase 1

(Received for publication, February 3, 1997, and in revised form, May 7, 1997)

From the Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark

The functionality of the sequence Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ of the substrate binding fourth - loop in the (/)₈-barrel of barley -amylase isozyme 1 (AMY1) was studied by random mutagenesis. A motif of polar Gly¹⁸⁴ hydrophobic residues was present in active mutants, selected by starch plate screening of yeast transformants. Gly¹⁸⁴ was important, probably due to the carbonyl group binding to Ca²⁺ and the spatial proximity of Phe¹⁸¹. Mutation of both flanking residues as in Ser¹⁸³-Gly¹⁸⁴-Met¹⁸⁵ (SGM-) and TGL-AMY1 decreased the Ca²⁺ affinity. SGM-AMY1 has 2-fold increased activity for amylose but reduced activity on maltooligosaccharides, whereas KGY-AMY1 has up to 3-fold elevated activity toward the oligosaccharides. TGL-AMY1 has modest activity on all substrates. Shifted action pattern on maltooligosaccharides for NGY-, SGM-, and TGL-AMY1 support that Arg¹⁸³ in wild type is located at subsites +1 and +2, accommodating two sugar rings toward the reducing end from the site of cleavage. In the crystal structure of barley -amylase 2 (AMY2), Lys¹⁸² (equivalent to AMY1 Arg¹⁸³) is hydrogen-bonded with sugar OH-3 in subsite +2. Higher K_i app for acarbose inhibition of KGY-AMY1 and parent AMY1 compared with the other mutants suggests favorable substrate interactions for Arg/Lys¹⁸³. KGY-AMY1 was not inhibited by the AMY2-specific proteinaceous barley -amylase/subtilisin inhibitor, although Lys¹⁸² of AMY2 is salt-linked to the inhibitor.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				X. Robert, R. Haser, H. Mori, B. Svensson, and N. Aghajari Oligosaccharide Binding to Barley {alpha}-Amylase 1 J. Biol. Chem., September 23, 2005; 280(38): 32968 - 32978. [Abstract] [Full Text] [PDF]

				E. Matsui, J. Abe, H. Yokoyama, and I. Matsui Aromatic Residues Located Close to the Active Center Are Essential for the Catalytic Reaction of Flap Endonuclease-1 from Hyperthermophilic Archaeon Pyrococcus horikoshii J. Biol. Chem., April 16, 2004; 279(16): 16687 - 16696. [Abstract] [Full Text] [PDF]

				K. S. Bak-Jensen, G. Andre, T. E. Gottschalk, G. Paes, V. Tran, and B. Svensson Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites -6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley {alpha}-Amylase 1 J. Biol. Chem., March 12, 2004; 279(11): 10093 - 10102. [Abstract] [Full Text] [PDF]

				M. H. Rivera, A. Lopez-Munguia, X. Soberon, and G. Saab-Rincon {alpha}-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity Protein Eng. Des. Sel., July 1, 2003; 16(7): 505 - 514. [Abstract] [Full Text] [PDF]

				E. Matsui, K. V. Musti, J. Abe, K. Yamasaki, I. Matsui, and K. Harata Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii J. Biol. Chem., September 27, 2002; 277(40): 37840 - 37847. [Abstract] [Full Text] [PDF]

				V. Monchois, M. Vignon, and R. R. B. Russell Mutagenesis of Asp-569 of Glucosyltransferase I Glucansucrase Modulates Glucan and Oligosaccharide Synthesis Appl. Envir. Microbiol., May 1, 2000; 66(5): 1923 - 1927. [Abstract] [Full Text]

				K. Ohdan, T. Kuriki, H. Kaneko, J. Shimada, T. Takada, Z. Fujimoto, H. Mizuno, and S. Okada Characteristics of Two Forms of alpha -Amylases and Structural Implication Appl. Envir. Microbiol., October 1, 1999; 65(10): 4652 - 4658. [Abstract] [Full Text]