STRUCTURAL CHANGES ON BINDING FAD AND FAD-ANALOGUES AND ON SITE-DIRECTED MUTAGENESIS OF GLUTATHIONE REDUCTASE

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg-138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

Download Full-text

Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human γ-glutamyl hydrolase

Biochemical Journal ◽

10.1042/bj3430551 ◽

1999 ◽

Vol 343 (3) ◽

pp. 551-555 ◽

Cited By ~ 5

Author(s):

Karen J. CHAVE ◽

John GALIVAN ◽

Thomas J. RYAN

Keyword(s):

Enzyme Activity ◽

Structural Changes ◽

Catalytic Mechanism ◽

Iodoacetic Acid ◽

Amino Acid Sequences ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Mutant Proteins ◽

Key Enzyme

γ-Glutamyl hydrolase (GH), which hydrolyses the γ-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

Download Full-text

Structural changes caused by site-directed mutagenesis of tyrosine-98 in Desulfovibrio vulgaris flavodoxin delineated by 1H and 15N NMR spectroscopy: implications for redox potential modulation

Biochemistry ◽

10.1021/bi00255a011 ◽

1994 ◽

Vol 33 (51) ◽

pp. 15298-15308 ◽

Cited By ~ 17

Author(s):

Brian J. Stockman ◽

Thomas E. Richardson ◽

Richard P. Swenson

Keyword(s):

Nmr Spectroscopy ◽

Redox Potential ◽

Structural Changes ◽

Site Directed Mutagenesis ◽

15N Nmr ◽

Desulfovibrio Vulgaris ◽

Directed Mutagenesis ◽

Desulfovibrio Vulgaris Flavodoxin ◽

15N Nmr Spectroscopy

Download Full-text

Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3090341 ◽

1995 ◽

Vol 309 (1) ◽

pp. 341-346 ◽

Cited By ~ 6

Author(s):

M H Rider ◽

K M Crepin ◽

M De Cloedt ◽

L Bertrand ◽

D Vertommen ◽

...

Keyword(s):

Structural Changes ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Kinetic Properties ◽

Wild Type ◽

Guanidinium Chloride ◽

Wild Type Enzyme ◽

Ph Profile ◽

Substrate Concentrations

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

Download Full-text

Crystal Structure and Desulfurization Mechanism of 2′-Hydroxybiphenyl-2-sulfinic Acid Desulfinase

Journal of Biological Chemistry ◽

10.1074/jbc.m602974200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32534-32539 ◽

Cited By ~ 32

Author(s):

Woo Cheol Lee ◽

Takashi Ohshiro ◽

Toshiyuki Matsubara ◽

Yoshikazu Izumi ◽

Masaru Tanokura

Keyword(s):

Crystal Structure ◽

Active Site ◽

Structural Changes ◽

Hydrophobic Interactions ◽

Rhodococcus Erythropolis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Active Site Residues ◽

Sulfinic Acid ◽

New Family

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2′-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

Download Full-text