scholarly journals A general kinetic approach to investigation of active-site availability in macromolecular catalysts

2000 ◽  
Vol 346 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Marina RESMINI ◽  
Sheraz GUL ◽  
Steve CARTER ◽  
Sanjiv SONKARIA ◽  
Christopher M. TOPHAM ◽  
...  
Keyword(s):  
Active Site ◽  
Kinetic Method ◽  
Order Rate Constant ◽  
Catalytic Antibody ◽  
Binding Model ◽  
Antibody Preparation ◽  
Single Turnover ◽  
Step Model ◽  
Catalytic Material ◽  
Antibody Preparations

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (Ea) and inert (i.e. non-binding, non-catalytic) material (Ei); (b) an extension of the conventional model (a) involving only Ea and Ei, but with non-productive binding to Ea (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (Eb), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters Vmax and Km obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (k, the limiting value of the first-order rate constant, kobs, at saturating concentrations of catalyst; and K) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack Eb, such as the non-productive binding model (b), may be calculated using [Ea]T = Vmax/k. This was validated by showing that, for α-chymotrypsin, identical values of [Ea]T were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known ‘all-or-none’ spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain Eb, such as polyclonal catalytic antibody preparations, Vmax/k is more complex, but provides an upper limit to [Ea]T. Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [Ea]T is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].

Kinetic and Titration Methods for Determination of Active Site Contents of Enzyme and Catalytic Antibody Preparations

Methods ◽  
2001 ◽  
Vol 24 (2) ◽  
pp. 153-167 ◽  
Author(s):  
Keith Brocklehurst ◽  
Marina Resmini ◽  
Christopher M. Topham
Keyword(s):  
Active Site ◽  
Catalytic Antibody ◽  
Antibody Preparations

scholarly journals Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

2013 ◽  
Vol 288 (22) ◽  
pp. 15532-15536 ◽  
Author(s):  
Natalie Gumataotao ◽  
Misty L. Kuhn ◽  
Natalia Hajnas ◽  
Richard C. Holz
Keyword(s):  
Active Site ◽  
Nitrile Hydratase ◽  
Stopped Flow ◽  
Single Turnover

scholarly journals Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

2003 ◽  
Vol 376 (3) ◽  
pp. 813-821 ◽  
Author(s):  
Sheraz GUL ◽  
Sanjiv SONKARIA ◽  
Surapong PINITGLANG ◽  
José FLOREZ-ALVAREZ ◽  
Syeed HUSSAIN ◽  
...  
Keyword(s):  
Structural Motif ◽  
Catalytic Antibody ◽  
Antibody Activity ◽  
Reaction Centre ◽  
Binding Characteristics ◽  
Leaving Group ◽  
Catalytic Mechanisms ◽  
Leaving Groups ◽  
Antibody Preparations ◽  
Ester Substrate

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in kcat/knon-cat and a 100-fold advantage in the proficiency constant, kcat/knon-cat·Km. Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

The Kinetic Basis of a General Method for the Investigation of Active Site Content of Enzymes and Catalytic Antibodies: First-Order Behaviour under Single-turnover and Cycling Conditions

2000 ◽  
Vol 204 (2) ◽  
pp. 239-256 ◽  
Author(s):  
CHRISTOPHER M. TOPHAM ◽  
SHERAZ GUL ◽  
MARINA RESMINI ◽  
SANJIV SONKARIA ◽  
GERRARD GALLACHER ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Antibodies ◽  
First Order ◽  
Single Turnover ◽  
General Method

In vitro Mutagenesis of Human Dihydropteridine Reductase at the Active Site and at Altered Sites Found in the Reductases of Deficient Children

Pteridines ◽  
1996 ◽  
Vol 7 (4) ◽  
pp. 123-136 ◽  
Author(s):  
Hong-Ping Zhang ◽  
Nan Yang ◽  
Wilfred L. F. Armarego
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
General Procedure ◽  
Maximum Velocity ◽  
Order Rate Constant ◽  
Site Directed Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Dihydropteridine Reductase ◽  
Proton Source

Summary A general procedure for in vitro site-directed mutagenesis of the wild-type dihydropteridine reductase gene has been used successfully to make eight mutant proteins. Five mutations were at the active site, viz Tyrl50His, Tyrl50Ser, Tyrl50Phe, Tyr150Glu and Tyrl50Lys. The proteins were expressed as glutathione S-transferase fusion proteins from which the unconjugated reductases were obtained by thrombin cleavage. The kinetic parameters of the conjugated and unconjugated reductases were measured using natural quinonoid R-7,8(6H)-dihydrobiopterin and non-natural quinonoid RS-6-methyl-7,8(6H)-dihydropterin and NADH. The kcat (maximum velocity at saturating concentrations of substrates) and kcatl Km (first order rate constant at low concentration of substrates) values show that the phenolic OH of Tyr 150 was the most likely proton source to complete the hydride reduction of the quinonoid pterin cofactor. However in the absence of a proton source at residue 150, measurable enzyme activities were observed indicating that a proton was relayed via a water molecule(s) from some neighbouring acidic amino acid residue. Three mutant dihydropteridine reductases, which were found in defective children, have been similarly attempted, viz GlylSlSer, Gly23Asp and a threonine insertion at position 123. The enzyme activities of the first two mutant reductases were consistent with the severity of the disease. The unconjugated reductase from the third mutation could not be obtained due to proteolysis but the fusion protein was enzymically active.

scholarly journals Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

scholarly journals 116 Polyclonal antibody preparations from avian origin improves growth performance of beef cattle during the first fourteen days of the backgrounding phase

2019 ◽  
Vol 97 (Supplement_1) ◽  
pp. 51-51
Author(s):  
Gleise Medeiros da Silva ◽  
Federico Podversich ◽  
Tessa Schulmeister ◽  
Ana C G Luna ◽  
Gonzalo Barreneche ◽  
...  
Keyword(s):  
Beef Cattle ◽  
Growth Performance ◽  
Polyclonal Antibody ◽  
Fusobacterium Necrophorum ◽  
Streptococcus Bovis ◽  
Feedlot Performance ◽  
Antibody Preparation ◽  
Orthogonal Contrasts ◽  
Avian Origin ◽  
Antibody Preparations

Abstract This study aimed to evaluate the effects of feeding avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (40, 35, and 25% of the preparation, respectively) on growth performance of beef cattle during the backgrounding phase. From d 0 to 56, Angus crossbreed heifers (n = 80; 360 ± 60 kg of BW; 470 ± 26 d of age) and steers (n = 20; 386 ± 65 kg of BW; 465 ± 30 d of age) were blocked by BW and randomly assigned to 1 of 16 concrete-floored pens (108 m2), equipped with 2 GrowSafe (GrowSafe Systems Ltd., Airdrie, Alberta, Canada) feed bunks each. Animals received a common ad libitum diet (76% TDN, 15.9% CP, DM basis) with the addition of 1 (PAP1), 3 (PAP3), or 0 g (CON) of PAP per d. Feed intake was recorded daily and BW were obtained on d -1, 0, 14, 28, 42, 55, and 56, to assess changes in BW, ADG, DMI, and G:F. Based upon orthogonal contrasts (CON vs. PAP1, and PAP1 vs. PAP3), BW and ADG on d 14, and DMI from 0 to 28, and 0 to 42 were greater for PAP1 vs. CON (P ≤ 0.03), whereas PAP3 animals were intermediate (P ≥ 0.20). No differences in final BW, DMI, and ADG from d 0 to 56 were detected among treatments (P ≥ 0.22). In conclusion, feeding 1g of polyclonal antibody preparations against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides in a backgrounding diet, improved growth performance in the first 14 d of feeding suggesting that feeding these PAP for longer than 14 d may not be necessary. The effects on subsequent feedlot performance when using PAP should be evaluated in future studies

scholarly journals Chloroacetone as an active-site-directed inhibitor of the aliphatic amidase from Pseudomonas aeruginosa

1980 ◽  
Vol 191 (3) ◽  
pp. 811-826 ◽  
Author(s):  
M R Hollaway ◽  
P H Clarke ◽  
T Ticho
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Chemical Reactivity ◽  
Covalent Bond ◽  
Order Rate Constant ◽  
Reaction Scheme ◽  
Enzymic Activity ◽  
Adsorption Complex ◽  
Apparent Dissociation Constant ◽  
Kinetic Experiment

1. Chloroacetone (I) was shown to be an active-site-directed inhibitor of the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa strain PAC142.2. This inhibitor reacted with the enzyme in two stages: the first involving the reversible formation of an enzymically inactive species, EI, and the second the formation of a species, EX, from which enzymic activity could not be recovered. 3. Different types of kinetic experiment were conducted to test conformity of the reaction to the scheme: E + I k+1 Equilibrium k-1 EI Leads to K+2 EX A computer-based analysis of the results was carried out and values of the individual rate constants were determined. 4. No direct evidence for a binding step before the formation of EI could be obtained, as with [E]0 Less Than [I]0 the observed first-order rate constant for the formation of EI was directly proportional to the concentration of chloroacetone up to 1.2 mM (above this concentration the reaction became too rapid to follow even by the stopped-flow method developed to investigate fast inhibition). 5. The value of k+1 exhibited a bell-shaped pH-dependency with a maximum value of about 3 × 10(3) M-1. S-1 at pH6 and apparent pKa values of 7.8 and about 4.8.6. The values of k-1 and K+2 were similar and changed with the time of reaction from values of about 3 × 10(-3) S-1 (pH8.6) at short times to about one-sixth this value for longer periods of incubation. In this respect the simple reaction scheme is insufficient to describe the inhibition process. 7. The overall inhibition reaction is rapid, whether it is considered in relation to the expected chemical reactivity of chloroacetone, the rate of reaction of other enzymes with substrate analogues containing the chloromethyl group, or the rate of the amidase-catalysed hydrolysis of N-methylacetamide, a substrate that is nearly isosteric with chloroacetone. 8. Acetamide protected the amidase from inhibition by chloroacetone, and the concentration-dependence of the protection gave a value of an apparent dissociation constant similar to the Km value for this substrate. 9. Addition of acetamide to solutions of the species EI led to a slow recovery of activity. Recovery of active enzyme was also observed after dilution of a solution of EI in the absence of substrate. 10. The species EI is considered not to be a simple adsorption complex, and the possibilities are discussed that it may be a tetrahedral carbonyl adduct, a Schiff base (azomethine) or a complex in which the enzyme has undergone a structural change. The species EX is probably a derivative in which there is a covalent bond between a group in the enzyme and the C-1 atom of the inhibitor.

Determination of the catalytic site content of a polyclonal catalytic antibody preparation

1998 ◽  
Vol 26 (2) ◽  
pp. S170-S170 ◽  
Author(s):  
MARINA RESMINI ◽  
SHERAZ GUL ◽  
SANJIV SONKARIA ◽  
GERARD GALLACHER ◽  
KEITH BROCKLEHURST
Keyword(s):  
Catalytic Site ◽  
Catalytic Antibody ◽  
Antibody Preparation

scholarly journals Structural evidence for a programmed general base in the active site of a catalytic antibody

2000 ◽  
Vol 97 (18) ◽  
pp. 9892-9895 ◽  
Author(s):  
B. Golinelli-Pimpaneau ◽  
O. Goncalves ◽  
T. Dintinger ◽  
D. Blanchard ◽  
M. Knossow ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Antibody ◽  
General Base
Sign in / Sign up

Export Citation Format

Share Document