scholarly journals The mechanism of addition of pyridoxal 5′-phosphate to Escherichia coli apo-serine hydroxymethyltransferase

2007 ◽  
Vol 404 (3) ◽  
pp. 477-485 ◽  
Author(s):  
Francesca Malerba ◽  
Andrea Bellelli ◽  
Alessandra Giorgi ◽  
Francesco Bossa ◽  
Roberto Contestabile
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Spectral Properties ◽  
Direct Evidence ◽  
Serine Hydroxymethyltransferase ◽  
Stopped Flow ◽  
Cofactor Binding ◽  
Kinetics Of ◽  
Flow Experiments ◽  
Step Mechanism

Previous studies suggest that the addition of pyridoxal 5′-phosphate to apo-serine hydroxymethyltransferase from Escherichia coli is the last event in the enzyme's folding process. We propose a mechanism for this reaction based on quenched-flow, stopped-flow and rapid-scanning stopped-flow experiments. All experiments were performed with an excess of apo-enzyme over cofactor, since excess pyridoxal 5′-phosphate results in a second molecule of cofactor binding to Lys346, which is part of the tetrahydropteroylglutamate-binding site. The equilibrium between the aldehyde and hydrate forms of the cofactor affects the kinetics of addition to the active site. Direct evidence of the formation of an intermediate aldimine between the cofactor and the active-site lysine was obtained. The results have been interpreted according to a three-step mechanism in which: (i) both aldehyde and hydrate forms of the cofactor bind rapidly and non-covalently to the apo-enzyme; (ii) only the aldehyde form reacts with the active-site lysine to give an intermediate internal aldimine with unusual spectral properties; and (iii) a final conformational change gives the native holo-enzyme.

scholarly journals Function of the active-site lysine in Escherichia coli serine hydroxymethyltransferase.

1993 ◽  
Vol 268 (31) ◽  
pp. 23132-23138
Author(s):  
D Schirch ◽  
S Delle Fratte ◽  
S Iurescia ◽  
S Angelaccio ◽  
R Contestabile ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Serine Hydroxymethyltransferase

THE TRYPSIN CATALYZED HYDROLYSIS OF p-NITROPHENYL ACETATE

1959 ◽  
Vol 37 (4) ◽  
pp. 751-759 ◽  
Author(s):  
James A. Stewart ◽  
Ludovic Ouellet
Keyword(s):  
Active Site ◽  
Initial Rate ◽  
Competitive Inhibition ◽  
Early Stage ◽  
Stopped Flow ◽  
Hydrogen Ions ◽  
Experimental Conditions ◽  
Influence Of Ph ◽  
Kinetics Of ◽  
Hydrolysis Of

The hydrolysis of p-nitrophenyl acetate (NPA) by trypsin has been investigated in the early stage of the reaction using stopped-flow techniques. The influence of pH on the initial rate suggests competitive inhibition of the active site of the enzyme by hydrogen ions. The dissociation constant of the enzyme obtained from the kinetics of this reaction (pK = 6.9) indicates possible catalysis by an ammo group or an imidazole group of the enzyme. Lysine methyl ester as an analogue of the enzyme catalyzes the hydrolysis of NPA under similar experimental conditions. The results are described in terms of an assumed mechanism and the nature of the catalytic site is discussed.

scholarly journals The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site

1980 ◽  
Vol 189 (3) ◽  
pp. 561-567 ◽  
Author(s):  
D Roberts ◽  
G L Kellett
Keyword(s):  
Skeletal Muscle ◽  
Cyclic Amp ◽  
Rate Constant ◽  
Catalytic Site ◽  
Stopped Flow ◽  
Diffusion Controlled ◽  
Inhibitory Site ◽  
Kinetics Of ◽  
Step Mechanism ◽  
Effector Binding

1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.

scholarly journals β-lactamase inhibitors. The inhibition of serine β-lactamases by specific boronic acids

1988 ◽  
Vol 251 (2) ◽  
pp. 453-459 ◽  
Author(s):  
I E Crompton ◽  
B K Cuthbert ◽  
G Lowe ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Boronic Acids ◽  
Penicillin G ◽  
Side Chain ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Step Mechanism

Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.

scholarly journals Stopped-flow spectrophotometric studies on the reaction of turkey liver xanthine dehydrogenase with reducing substrates

1978 ◽  
Vol 171 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Í N Fhaoláin ◽  
M J Hynes ◽  
M P Coughlan
Keyword(s):  
Active Site ◽  
Rate Constants ◽  
Xanthine Dehydrogenase ◽  
Stopped Flow ◽  
Redox Potentials ◽  
First Order ◽  
The Third ◽  
Enzyme Functionality ◽  
Kinetics Of ◽  
Kinetics Of Reduction

The kinetics of reduction of turkey liver xanthine dehydrogenase by substrates were investigated by stopped-flow spectrophotometry. The results may be explained in terms of the known redox potentials of the various centres in the enzyme [Barber, Bray, Cammack & Coughlan (1977) Biochem. J. 163, 279-289]. They are, morover, consistent with the scheme [Olson, Ballou, Palmer & Massey (1974) J. Biol. Chem. 249, 4363-4382] in which reduction occurs in three consecutive steps, one molecule of substrate reacting with the active site at each step. First-order rate constants believed to correspond respectively to the combined first and second steps and to the third step in the reduction by excess of xanthine and of NADH were determined. The rates of reaction with these substrates in the combined first and second steps are independent of the degree of enzyme functionality.

Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase - the role of evolutionarily conserved hydrophobic contacts

FEBS Journal ◽  
2009 ◽  
Vol 276 (24) ◽  
pp. 7319-7328 ◽  
Author(s):  
Rita Florio ◽  
Roberta Chiaraluce ◽  
Valerio Consalvi ◽  
Alessandro Paiardini ◽  
Bruno Catacchio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Structural Stability ◽  
Serine Hydroxymethyltransferase ◽  
Cofactor Binding ◽  
Evolutionarily Conserved

scholarly journals Kinetic characterization of the membrane-bound cytochromes of Escherichia coli grown under a variety of conditions by using a stopped-flow dual-wavelength spectrophotometer

1976 ◽  
Vol 154 (2) ◽  
pp. 285-294 ◽  
Author(s):  
B A. Haddock ◽  
J. A Downie ◽  
P B. Garland
Keyword(s):  
Escherichia Coli ◽  
Oxidation Kinetics ◽  
Difference Spectrum ◽  
Stopped Flow ◽  
Oxidation Rates ◽  
Cytochrome O ◽  
Membrane Bound ◽  
Cytochrome D ◽  
Kinetics Of

A study was made of the rapid oxidation kinetics of the cytochromes of Escherichia coli. The b-type cytochromes were kinetically heterogeneous, with one species (presumably cytochrome o) oxidized so rapidly that it could fully support observed oxidation rates. Cytochrome d but not cytochrome a1 was also kinetically competent to support respiration. However, in cells grown anaerobically in the presence of NO3-, cytochrome d exhibited slow oxidation kinetics and a red-shift in its reduced-minus-oxidized difference spectrum.

scholarly journals Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca2+ and of Desensitization by Trypsin

2003 ◽  
Vol 185 (14) ◽  
pp. 4233-4242 ◽  
Author(s):  
Athena Sudom ◽  
Robert Walters ◽  
Landon Pastushok ◽  
Douglas Goldie ◽  
Lata Prasad ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Catalytic Mechanism ◽  
Stop Codon ◽  
Allosteric Site ◽  
C Terminus ◽  
Allosteric Regulator ◽  
Kinetics Of ◽  
Resolution Structure

ABSTRACT The 1.8-Å resolution structure of the ATP-Mg2+-Ca2+-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg2+-Mn2+-pyruvate-PCK, except for the Ca2+ and Mn2+ binding sites. Ca2+ was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca2+ bound only at the active site, indicating that there is likely no surface allosteric site. 45Ca2+ bound to PCK with a K d of 85 μM and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca2+. Separate roles of Mg2+, which binds the nucleotide, and Ca2+, which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca2+-bound structure. Partial trypsin digestion abolishes Ca2+ activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca2+ binding site, probably stabilizing the C terminus. Phe409Ala, ΔPhe409, Phe413Ala, Δ397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca2+ site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.

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