HlyC, the Internal Protein Acyltransferase That Activates Hemolysin Toxin:  The Role of Conserved Tyrosine and Arginine Residues in Enzymatic Activity As Probed by Chemical Modification and Site-Directed Mutagenesis†

Biochemistry ◽  
1999 ◽  
Vol 38 (27) ◽  
pp. 8831-8838 ◽  
Author(s):  
M. Stephen Trent ◽  
Lesa M. S. Worsham ◽  
M. Lou Ernst-Fonberg
Keyword(s):  
Chemical Modification ◽  
Enzymatic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Internal Protein ◽  
Arginine Residues ◽  
Conserved Tyrosine

scholarly journals Point mutations of two arginine residues in the Streptomyces R61 dd-peptidase

1992 ◽  
Vol 283 (1) ◽  
pp. 123-128 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Ghuysen ◽  
J M Frère
Keyword(s):  
Enzyme Activity ◽  
Enzyme Stability ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Arginine Residues

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

Role of lysines in human angiogenin: chemical modification and site-directed mutagenesis

Biochemistry ◽  
1989 ◽  
Vol 28 (4) ◽  
pp. 1726-1732 ◽  
Author(s):  
Robert Shapiro ◽  
Edward A. Fox ◽  
James F. Riordan
Keyword(s):  
Chemical Modification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

2008 ◽  
Vol 410 (3) ◽  
pp. 543-553 ◽  
Author(s):  
Prashanth S. Jois ◽  
Nagaraj Madhu ◽  
Desirazu N. Rao
Keyword(s):  
Chemical Modification ◽  
Dna Methyltransferase ◽  
Catalytic Mechanism ◽  
Gel Filtration ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Recognition Sequence ◽  
Native Page ◽  
Histidine Residues

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5′-CAGCAG-3′. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.

scholarly journals Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

1991 ◽  
Vol 275 (2) ◽  
pp. 447-452 ◽  
Author(s):  
M Lander ◽  
A R Pitt ◽  
P R Alefounder ◽  
D Bardy ◽  
C Abell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Enzyme Substrate ◽  
Arginine Residues

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

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