SITE-SPECIFIC MUTAGENESIS OF THE METAL-BINDING SITES OF PORCINE FRUCTOSE-1,6-BISPHOSPHATASE

被引:14
作者
CHEN, LR [1 ]
HEGDE, R [1 ]
CHEN, M [1 ]
FROMM, HJ [1 ]
机构
[1] IOWA STATE UNIV SCI & TECHNOL, DEPT BIOCHEM & BIOPHYS, AMES, IA 50011 USA
关键词
D O I
10.1006/abbi.1993.1599
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mutations in the metal binding sites of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme. The mutant and wild-type enzymes have been characterized by circular dichroism spectrometry and initial-rate kinetics. One of the mutant forms of fructose-1,6-bisphosphatase (Glu280Gln) is associated with a single metal site, whereas two other mutants (Glu97Gln and Asp118Asn) ligate two metal ions. The mutant enzymes exhibit very large decreases in kcat relative to the wild-type enzyme; however, other kinetic parameters, such as Km values, are not greatly altered. Metal binding cooperativity and binding affinity is decreased in the mutants compared to wild-type fructose-1,6-bisphosphatase. Mutations in the metal binding sites greatly enhance the enzyme′s affinity for AMP, a potent regulator of fructose-1,6-bisphosphatase activity. The results of these investigations are fully consistent with predictions made on the role of specific amino acid residues at the metal binding sites in porcine fructose-1,6-bisphosphatase from X-ray diffraction studies [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N. (1993) Biochemistry, 32, 1844-1857]. © 1993 Academic Press, Inc.
引用
收藏
页码:350 / 354
页数:5
相关论文
共 34 条
[1]   BINDING AND KINETIC DATA FOR RABBIT LIVER FRUCTOSE-1,6-BISPHOSPHATASE WITH ZN2+ AS COFACTOR [J].
BENKOVIC, PA ;
CAPERELLI, CA ;
DEMAINE, M ;
BENKOVIC, SJ .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1978, 75 (05) :2185-2189
[2]  
BENKOVIC SJ, 1982, ADV ENZYMOL RAMB, V53, P45
[3]   HIGH-LEVEL EXPRESSION OF PORCINE FRUCTOSE-1,6-BISPHOSPHATASE IN ESCHERICHIA-COLI - PURIFICATION AND CHARACTERIZATION OF THE ENZYME [J].
BURTON, VA ;
CHEN, M ;
ONG, WC ;
LING, TT ;
FROMM, HJ ;
STAYTON, MM .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1993, 192 (02) :511-517
[4]  
DUDMAN NPB, 1978, J BIOL CHEM, V253, P5712
[5]  
ELMAGHRABI MR, 1992, J BIOL CHEM, V267, P6526
[6]  
ELMAGHRABI MR, 1993, J BIOL CHEM, V268, P9466
[7]  
GANSON NJ, 1985, J BIOL CHEM, V260, P2837
[8]  
Gomori G, 1943, J BIOL CHEM, V148, P139
[9]   FRUCTOSE-BISPHOSPHATASE AS A SUBSTRATE OF CYCLIC AMP-DEPENDENT PROTEIN-KINASE [J].
HOSEY, MM ;
MARCUS, F .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1981, 78 (01) :91-94
[10]   STRUCTURE REFINEMENT OF FRUCTOSE-1,6-BISPHOSPHATASE AND ITS FRUCTOSE 2,6-BISPHOSPHATE COMPLEX AT 2.8-A RESOLUTION [J].
KE, H ;
THORPE, CM ;
SEATON, BA ;
LIPSCOMB, WN ;
MARCUS, F .
JOURNAL OF MOLECULAR BIOLOGY, 1990, 212 (03) :513-539