scholarly journals Kinetics of protein modification reactions. Plot of fractional enzyme activity versus extent of protein modification in cases where all modifiable groups are essential for enzyme activity

1984 ◽  
Vol 223 (1) ◽  
pp. 259-262 ◽  
Author(s):  
E T Rakitzis
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Protein Modification ◽  
Enzyme Molecule ◽  
Single Group ◽  
Initial Portion ◽  
Enzyme Active Site ◽  
Kinetics Of

The plot of fractional enzyme activity versus extent of protein modification, for cases where all enzyme modifiable groups of a certain kind are essential for activity, is found to be nearly independent of the number, per enzyme active site, of modifiable groups involved. Such plots usually, by a fallacious extension of the initial portion of the plot on the extent-of-modification axis, are interpreted to mean the modification of one single group per enzyme active site (or per enzyme molecule). The possible relevance of these findings to cases in the literature is discussed.

scholarly journals α-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin

1980 ◽  
Vol 187 (2) ◽  
pp. 345-352 ◽  
Author(s):  
N I Tarasova ◽  
G I Lavrenova ◽  
V M Stepanov
Keyword(s):  
Enzyme Activity ◽  
Carboxy Group ◽  
Active Site ◽  
Protein Modification ◽  
Phenacyl Bromide ◽  
Enzyme Molecule ◽  
Noticeable Effect ◽  
Porcine Pepsin ◽  
Enzyme Active Site ◽  
Phenacyl Bromides

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.

A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity

2008 ◽  
Vol 389 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Jian-Qiang Fan
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Protein Misfolding ◽  
Enzyme Inhibitors ◽  
Lysosomal Storage ◽  
New Paradigm ◽  
Inherited Disorders ◽  
Pharmacological Chaperone ◽  
Mutant Proteins ◽  
Enzyme Active Site

Abstract Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.

scholarly journals Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens

2000 ◽  
Vol 345 (2) ◽  
pp. 315-319 ◽  
Author(s):  
Dieter BECKER ◽  
Karin S. H. JOHNSON ◽  
Anu KOIVULA ◽  
Martin SCHÜLEIN ◽  
Michael L. SINNOTT
Keyword(s):  
Trichoderma Reesei ◽  
Active Site ◽  
Substrate Concentration ◽  
Initial Rate ◽  
Enzyme Active Site ◽  
Humicola Insolens ◽  
Cellobiohydrolase Ii ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Identical Crystal

We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

scholarly journals Kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline

1992 ◽  
Vol 281 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Z X Wang ◽  
H B Wu ◽  
X C Wang ◽  
H M Zhou ◽  
C L Tsou
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Inactivation Kinetics ◽  
Slow Inactivation ◽  
Phenanthroline Complex ◽  
Rapid Reaction ◽  
Kinetics Of ◽  
Active Site Conformation ◽  
Inhibited Enzyme ◽  
Substrate Competition

The kinetic theory of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] has been applied to a study on the kinetics of the course of inactivation of aminoacylase by 1,10-phenanthroline. Upon dilution of the enzyme that had been incubated with 1,10-phenanthroline into the reaction mixture, the activity of the inhibited enzyme gradually increased, indicating dissociation of a reversible enzyme–1,10-phenanthroline complex. The kinetics of the substrate reaction with different concentrations of the substrate chloroacetyl-L-alanine and the inactivator suggest a complexing mechanism for inactivation by, and substrate competition with, 1,10-phenanthroline at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-Zn(2+)-1,10-phenanthroline complex is a relatively rapid reaction, followed by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.

Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H2O2 and OH radicals

1976 ◽  
Vol 54 (2) ◽  
pp. 242-253 ◽  
Author(s):  
Wen Shu Lin ◽  
G. Maurice Gaucher ◽  
David A. Armstrong ◽  
Manohar Lal
Keyword(s):  
Hydrogen Peroxide ◽  
Computer Program ◽  
Active Site ◽  
Proteolytic Enzyme ◽  
Oh Radicals ◽  
Sulfenic Acid ◽  
Enzyme Active Site ◽  
Mixed Disulfide ◽  
Kinetics Of ◽  
Saturated Solutions

The inactivation of the proteolytic enzyme papain by hydrogen peroxide produces a sulfenic acid by oxidation of the essential SH of cysteine 25 at the enzyme active site:[Formula: see text]The kinetics of repair of this entity by cysteine were consistent with the two reactions:[Formula: see text][Formula: see text]Reaction 4 was the faster with k4 ≥ 800 M−1 s−1, and k5 = 11.3 ± 0.5 M−1 s−1. A computer program was developed to evaluate the contributions of peroxide-inactivation and cysteine-repair when they occur simultaneously in N2O-saturated solutions in the absence of catalase. The yields predicted by this program agreed well with the inactivation caused by peroxide in irradiated systems.The effect of cysteine on the inactivation of papain by OH radicals produced by radiolysis of N2O-saturated solutions containing catalase was also investigated. Protection against permanent inactivation was much more efficient than expected on the basis of a simple competition between cysteine and papain for OH radicals, but there was a marked increase in the yield of repairable damage which was not due to hydrogen peroxide. These observations can be qualitatively accounted for by the reactions:[Formula: see text]The same rate constant was obtained for the repair of PapainCys25SSCys from this source as from the peroxide inactivation and treatment with cysteine. However, there was also evidence for additional cysteine-sensitized production of mixed disulfide and this probably occurs through reactions of CysS• radicals:[Formula: see text]

scholarly journals The molecular basis of the effect of temperature on enzyme activity

2009 ◽  
Vol 425 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Roy M. Daniel ◽  
Michelle E. Peterson ◽  
Michael J. Danson ◽  
Nicholas C. Price ◽  
Sharon M. Kelly ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Classical Model ◽  
Active Form ◽  
Enzyme Reaction ◽  
Effect Of Temperature ◽  
Enzyme Active Site ◽  
Temperature Ranges ◽  
The Difference ◽  
Two State Model

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (ΔHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of ΔG*cat made on the basis of the two-state (‘Classical’) model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

scholarly journals The Organ Distribution, Characterization and Modification of Acetylcholinesterase Activity in Adult African Grasshopper: Zonocerus sp Linn.

2019 ◽  
pp. 1-9
Author(s):  
E. A. Fajemisin ◽  
O. S. Bamidele ◽  
S. O. Ogunsola ◽  
E. A. Aiyenuro
Keyword(s):  
Enzyme Activity ◽  
Protein Content ◽  
Active Site ◽  
Ache Activity ◽  
Specific Activity ◽  
Acetylcholinesterase Activity ◽  
Organ Distribution ◽  
Enzyme Active Site ◽  
University Of Technology ◽  
Tryptophan Residues

Aim: To determine the organ distribution and characterization of acetylcholinesterase in the adult African variegated grasshoppers – Zonocerus variegatus and Zonocerus elegans. (Zonocerus Sp. Linn) Place and Duration of the Study: The insect model: African variegated grasshoppers are gotten from the Open green fields at the Federal University of Technology, Akure, Nigeria, and research was carried out between March and June, 2016 in the Enzymology laboratory, Biochemistry department, Federal University of Technology, Akure, Nigeria. Methodology: Twenty (20) adults variegated grasshoppers were taken from the Open field in the University community, and taken to the Biology department for Identification. After identification, the specimen was weighed, freeze, dissected into fractions (Head, Thorax and Abdomen) and then homogenized to get the crude protein extract. The crude enzyme extract is further purified using the Ion-exchange chromatography with column bed packed with DEAE – Sephadex A50. The protein content of the purified AChE was determined using the Lowry method while the Acetylcholinesterase activity was determined by the Ellman’s assay procedures. The characterization of AChE was tested by modifying agent such as N-Bromo Succinamide (NBS) which confirms the presence of key aromatic proteins involve in catalysis at the active site of the enzyme. Results: The protein concentration according to their fractions: Head (35.7%), Thorax (29.2%), and Abdomen (35.1%). The AChE activity according to their fractions: Head (38.6%), Thorax (23.7%), and Abdomen (37.7%). The specific activity which relates the AChE activity to protein content is given: Head (28.8%), Thorax (40.4%), and Abdomen (30.8%). From the Organ distribution and AChE activity, it was observed that the Head Fractions has the Highest protein content, and Enzyme activity. Comparatively, there are slight differences in the Enzyme activity of the Head and Abdominal fractions which represents the two peaks in the AChE chart. As well, the thorax has the highest specific activity. The modification by the chemical agent NBS shows a drastic decrease (about 50%) in Enzyme activity and characterize enzyme active site with aromatic proteins especially tryptophan residues. Conclusion: Research findings shows the dominance of AChE protein in the Head region, hence high enzyme activity (useful for nervous coordination) as well as presence of tryptophan residues at the enzyme active site. The importance of research is useful in enzymology, neuroscience and public health.

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