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 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 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 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 Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors

2005 ◽  
Vol 392 (1) ◽  
pp. 103-116 ◽  
Author(s):  
Baruch Karniol ◽  
Jeremiah R. Wagner ◽  
Joseph M. Walker ◽  
Richard D. Vierstra
Keyword(s):  
Red Light ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Genomic Analyses ◽  
Biochemical Studies ◽  
Lyase Activity ◽  
Acidic Environments ◽  
Micro Organisms

Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659–3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

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.

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