scholarly journals Mutations within a highly conserved sequence present in the X region of phosphoinositide-specific phospholipase C-δ1

1995 ◽  
Vol 307 (1) ◽  
pp. 69-75 ◽  
Author(s):  
M V Ellis ◽  
U Sally ◽  
M Katan
Keyword(s):  
Enzyme Activity ◽  
Phospholipase C ◽  
Limited Proteolysis ◽  
Structural Similarity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Histidine Residues ◽  
Conserved Sequence ◽  
Hydrolysis Of

Phosphoinositide-specific phospholipase C (PI-PLC) enzymes have considerable structural similarity within limited regions (X and Y) implicated in catalysis. The role of residues contained within a highly conserved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta 1 isoenzyme. Seven residues (Ser-308, Ser-309, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residues, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme activity. The kcat of hydrolysis of phosphatidylinositol 4,5-bisphosphate by H311A and Y314A mutants was reduced 1000- and 10-fold respectively, with little effect on Km. Further analysis of H311A and Y314A mutants, using limited proteolysis and circular dichroism, had shown that no major structural alterations had occurred. Since site-directed mutagenesis demonstrated the importance of histidine residues, their role in enzyme function was also analysed by chemical modification with diethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.

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 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.

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