scholarly journals Effect of Site-Directed Mutagenesis of Methylglyoxal-Modifiable Arginine Residues on the Structure and Chaperone Function of Human αA-Crystallin†

Biochemistry ◽  
2006 ◽  
Vol 45 (14) ◽  
pp. 4569-4577 ◽  
Author(s):  
Ashis Biswas ◽  
Antonia Miller ◽  
Tomoko Oya-Ito ◽  
Puttur Santhoshkumar ◽  
Manjunatha Bhat ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chaperone Function ◽  
Arginine Residues

scholarly journals Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2

2002 ◽  
Vol 366 (3) ◽  
pp. 971-976 ◽  
Author(s):  
Lucimara CHIOATO ◽  
Arthur H.C. de OLIVEIRA ◽  
Roberto RULLER ◽  
Juliana M. SÁ ◽  
Richard J. WARD
Keyword(s):  
Phospholipase A2 ◽  
Lipid Membrane ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Phospholipid Bilayer ◽  
Directed Mutagenesis ◽  
Aromatic Residues ◽  
Loop Region ◽  
Terminal Loop ◽  
Arginine Residues

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding

1995 ◽  
Vol 312 (2) ◽  
pp. 357-365 ◽  
Author(s):  
K H Mayo ◽  
E Ilyina ◽  
V Roongta ◽  
M Dundas ◽  
J Joseph ◽  
...  
Keyword(s):  
Alpha Helix ◽  
Platelet Factor 4 ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Heparin Binding ◽  
Nmr Studies ◽  
Platelet Factor ◽  
Binding Data ◽  
Arginine Residues ◽  
Proton Resonances

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.

Site-Directed Mutagenesis of Human Prorenin. Substitution of Three Arginine Residues in the Propeptide with Glutamine Residues Yields Active Prorenin1

1990 ◽  
Vol 107 (1) ◽  
pp. 27-31 ◽  
Author(s):  
Takeshi Yamauchi ◽  
Masami Nagahama ◽  
Toshio Watanabe ◽  
Yasuyuki Ishizuka ◽  
Hitoshi Hori ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Arginine Residues

scholarly journals Effect of site-directed mutagenesis of the arginine residues 509 and 748 on mouse band 3 protein-mediated anion transport

1998 ◽  
Vol 1371 (1) ◽  
pp. 114-122 ◽  
Author(s):  
Doris Karbach ◽  
Martin Staub ◽  
Phillip G Wood ◽  
Hermann Passow
Keyword(s):  
Band 3 ◽  
Anion Transport ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Band 3 Protein ◽  
Arginine Residues

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.

Catalytic Roles of Arginine Residues 82 and 92 ofEscherichia coli6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase:  Site-Directed Mutagenesis and Biochemical Studies†

Biochemistry ◽  
2003 ◽  
Vol 42 (6) ◽  
pp. 1581-1588 ◽  
Author(s):  
Yue Li ◽  
Yan Wu ◽  
Jaroslaw Blaszczyk ◽  
Xinhua Ji ◽  
Honggao Yan
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Biochemical Studies ◽  
Arginine Residues

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.

scholarly journals Critical role of Arg433 in rat transketolase activity as probed by site-directed mutagenesis

1998 ◽  
Vol 333 (2) ◽  
pp. 367-372 ◽  
Author(s):  
Yunjo SOH ◽  
Byoung J. SONG ◽  
Jiingjau JENG ◽  
Abraham T. KALLARAKAL
Keyword(s):  
Critical Role ◽  
Immunoblot Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Hydrogen Bond Interactions ◽  
Apparent Homogeneity ◽  
Arginine Residues ◽  
Biochemical Analyses

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405–2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102 → Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350 → Ala, Arg506 → Ala and Arg433 → Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350 → Ala and Arg433 → Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433 → Ala mutant protein was less stable than the wild-type and Arg350 → Ala proteins at 55 °C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433 → Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433 → Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

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