Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by carbon-13 NMR and correlation with kinetic studies

Biochemistry ◽  
1989 ◽  
Vol 28 (5) ◽  
pp. 2116-2124 ◽  
Author(s):  
Charles B. Grissom ◽  
John L. Markley
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Staphylococcal Nuclease

scholarly journals Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes

1986 ◽  
Vol 234 (2) ◽  
pp. 343-347 ◽  
Author(s):  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

1995 ◽  
Vol 308 (3) ◽  
pp. 1017-1023 ◽  
Author(s):  
I P Street ◽  
S G Withers
Keyword(s):  
Active Site ◽  
Glycogen Phosphorylase ◽  
Ph Dependence ◽  
Substrate Inhibition ◽  
19F Nmr ◽  
Ionization State ◽  
Nmr Studies ◽  
Substrate Analogues ◽  
Glycogen Phosphorylase B ◽  
Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

scholarly journals The Interaction of Nucleotides with the Active Site of Staphylococcal Nuclease

1967 ◽  
Vol 242 (20) ◽  
pp. 4759-4767 ◽  
Author(s):  
Pedro Cuatrecasas ◽  
Sara Fuchs ◽  
Christian B. Anfinsen
Keyword(s):  
Active Site ◽  
Staphylococcal Nuclease

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

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