scholarly journals New standards for collecting and fitting steady state kinetic data

2019 ◽  
Vol 15 ◽  
pp. 16-29 ◽  
Author(s):  
Kenneth A Johnson
Keyword(s):  
Conformational Changes ◽  
Short Review ◽  
Order Rate Constant ◽  
Rate Equations ◽  
Enzyme Specificity ◽  
Additional Information ◽  
Steady State Kinetic ◽  
Specificity Constant ◽  
State Kinetic

The Michaelis–Menten equation is usually expressed in terms of k cat and K m values: v = k cat[S]/(K m + [S]). However, it is impossible to interpret K m in the absence of additional information, while the ratio of k cat/K m provides a measure of enzyme specificity and is proportional to enzyme efficiency and proficiency. Moreover, k cat/K m provides a lower limit on the second order rate constant for substrate binding. For these reasons it is better to redefine the Michaelis–Menten equation in terms of k cat and k cat/K m values: v = k SP[S]/(1 + k SP[S]/k cat), where the specificity constant, k SP = k cat/K m. In this short review, the rationale for this assertion is explained and it is shown that more accurate measurements of k cat/K m can be derived directly using the modified form of the Michaelis–Menten equation rather than calculated from the ratio of k cat and K m values measured separately. Even greater accuracy is achieved with fitting the raw data directly by numerical integration of the rate equations rather than using analytically derived equations. The importance of fitting to derive k cat and k cat/K m is illustrated by considering the role of conformational changes in enzyme specificity where k cat and k cat/K m can reflect different steps in the pathway. This highlights the pitfalls in attempting to interpret K m, which is best understood as the ratio of k cat divided by k cat/K m.

scholarly journals Role of Homoserine Transacetylase as a New Target for Antifungal Agents

2007 ◽  
Vol 51 (5) ◽  
pp. 1731-1736 ◽  
Author(s):  
Ishac Nazi ◽  
Adam Scott ◽  
Anita Sham ◽  
Laura Rossi ◽  
Peter R. Williamson ◽  
...  
Keyword(s):  
Small Molecules ◽  
Antifungal Agents ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Small Molecule Inhibition ◽  
Steady State Kinetic ◽  
Antibiotic Discovery ◽  
Homoserine Transacetylase ◽  
State Kinetic

ABSTRACT Microbial amino acid biosynthesis is a proven yet underexploited target of antibiotics. The biosynthesis of methionine in particular has been shown to be susceptible to small-molecule inhibition in fungi. The first committed step in Met biosynthesis is the acylation of homoserine (Hse) by the enzyme homoserine transacetylase (HTA). We have identified the MET2 gene of Cryptococcus neoformans H99 that encodes HTA (CnHTA) by complementation of an Escherichia coli metA mutant that lacks the gene encoding homoserine transsuccinylase (HTS). We cloned, expressed, and purified CnHTA and determined its steady-state kinetic parameters for the acetylation of L-Hse by acetyl coenzyme A. We next constructed a MET2 mutant in C. neoformans H99 and tested its growth behavior in Met-deficient media, confirming the expected Met auxotrophy. Furthermore, we used this mutant in a mouse inhalation model of infection and determined that MET2 is required for virulence. This makes fungal HTA a viable target for new antibiotic discovery. We screened a 1,000-compound library of small molecules for HTA inhibitors and report the identification of the first inhibitor of fungal HTA. This work validates HTA as an attractive drug-susceptible target for new antifungal agent design.

scholarly journals Efficient catalysis by β-lactamase from Staphylococcus aureus PC1 accompanied by accumulation of an acyl-enzyme

1996 ◽  
Vol 315 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Xiaolin QI ◽  
Richard VIRDEN
Keyword(s):  
Steady State ◽  
Blue Shift ◽  
Order Rate Constant ◽  
Diffusion Limited ◽  
Steady State Kinetic ◽  
Non Covalent Interactions ◽  
Ph Range ◽  
State Kinetic ◽  
Covalent Interactions ◽  
Viscosity Dependence

The pH- and temperature-dependence of steady-state kinetic parameters for 6-β-(2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 β-lactamase, and the viscosity-dependence of Km/kcat indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4–9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to kcat. The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation.

scholarly journals Unravelling ceftazidime/avibactam resistance of KPC-28, a KPC-2 variant lacking carbapenemase activity

2019 ◽  
Vol 74 (8) ◽  
pp. 2239-2246 ◽  
Author(s):  
Saoussen Oueslati ◽  
Bogdan I Iorga ◽  
Linda Tlili ◽  
Cynthia Exilie ◽  
Agustin Zavala ◽  
...  
Keyword(s):  
Amino Acid ◽  
Susceptibility Testing ◽  
Fold Increase ◽  
Hydrolytic Activity ◽  
E Coli ◽  
Steady State Kinetic ◽  
Double Amino Acid ◽  
Or Gene ◽  
State Kinetic

Abstract Background KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions. Objectives To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243). Methods The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity. Results Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate. Conclusions We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection.

Is the single transition-state model appropriate for the fundamental reactions of organic chemistry?

2005 ◽  
Vol 77 (11) ◽  
pp. 1823-1833 ◽  
Author(s):  
Vernon D. Parker
Keyword(s):  
Steady State ◽  
Kinetic Method ◽  
Order Rate Constant ◽  
Product Formation ◽  
Second Order ◽  
Excess Reagent ◽  
First Order ◽  
Steady State Kinetic ◽  
Pseudo First Order ◽  
State Kinetic

In recent years, we have reported that a number of organic reactions generally believed to follow simple second-order kinetics actually follow a more complex mechanism. This mechanism, the reversible consecutive second-order mechanism, involves the reversible formation of a kinetically significant reactant complex intermediate followed by irreversible product formation. The mechanism is illustrated for the general reaction between reactant and excess reagent under pseudo-first-order conditions in eq. i where kf' is the pseudo-first-order rate constant equal to kf[Excess Reagent].Reactant + Excess reagent = Reactant complex = Products (i)The mechanisms are determined for the various systems, and the kinetics of the complex mechanisms are resolved by our "non-steady-state kinetic data analysis". The basis for the non-steady-state kinetic method will be presented along with examples. The problems encountered in attempting to identify intermediates formed in low concentration will be discussed.

Recognition of aminoglycoside antibiotics by enterococcal-staphylococcal aminoglycoside 3'-phosphotransferase type IIIa: role of substrate amino groups.

1996 ◽  
Vol 40 (11) ◽  
pp. 2648-2650 ◽  
Author(s):  
G A McKay ◽  
J Roestamadji ◽  
S Mobashery ◽  
G D Wright
Keyword(s):  
Steady State ◽  
Aminoglycoside Antibiotics ◽  
Hydroxyl Group ◽  
Amino Group ◽  
Amino Groups ◽  
Type Iiia ◽  
Steady State Kinetic ◽  
State Kinetic

The interactions of the aminoglycoside 3'-phosphotransferase IIIa with aminoglycoside antibiotics lacking specific amino groups were examined by steady-state kinetic analyses. The results demonstrate that an amino group on C-1 and either an amino or a hydroxyl group at the 2' and 6' positions are important for detoxification of aminoglycosides by this enzyme.

Role of spectrin in membrane fusion and in shape change of human erythrocyte ghost

1978 ◽  
Vol 36 (2) ◽  
pp. 328-329
Author(s):  
Hideo Hayashi ◽  
Yoshikazu Hirai ◽  
John T. Penniston
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Shape Change ◽  
Membrane Surface ◽  
Slight Modification ◽  
Active Role ◽  
Main Role ◽  
Bank Blood ◽  
Human Erythrocyte Ghost

Spectrin is a membrane associated protein most of which properties have been tentatively elucidated. A main role of the protein has been assumed to give a supporting structure to inside of the membrane. As reported previously, however, the isolated spectrin molecule underwent self assemble to form such as fibrous, meshwork, dispersed or aggregated arrangements depending upon the buffer suspended and was suggested to play an active role in the membrane conformational changes. In this study, the role of spectrin and actin was examined in terms of the molecular arrangements on the erythrocyte membrane surface with correlation to the functional states of the ghosts.Human erythrocyte ghosts were prepared from either freshly drawn or stocked bank blood by the method of Dodge et al with a slight modification as described before. Anti-spectrin antibody was raised against rabbit by injection of purified spectrin and partially purified.

POPDC proteins and cardiac function

2019 ◽  
Vol 47 (5) ◽  
pp. 1393-1404 ◽  
Author(s):  
Thomas Brand
Keyword(s):  
Potential Role ◽  
Striated Muscle ◽  
Short Review ◽  
Effector Proteins ◽  
Muscle Disease ◽  
Disease Genes ◽  
Protein Protein Interaction ◽  
Interaction Partners ◽  
And Function

Abstract The Popeye domain-containing gene family encodes a novel class of cAMP effector proteins in striated muscle tissue. In this short review, we first introduce the protein family and discuss their structure and function with an emphasis on their role in cyclic AMP signalling. Another focus of this review is the recently discovered role of POPDC genes as striated muscle disease genes, which have been associated with cardiac arrhythmia and muscular dystrophy. The pathological phenotypes observed in patients will be compared with phenotypes present in null and knockin mutations in zebrafish and mouse. A number of protein–protein interaction partners have been discovered and the potential role of POPDC proteins to control the subcellular localization and function of these interacting proteins will be discussed. Finally, we outline several areas, where research is urgently needed.

Role of the Structural Characteristics of Dendrimers in the Manifestation of the Antiamyloid Properties

INEOS OPEN ◽  
2020 ◽  
Vol 3 ◽  
Author(s):  
S. A. Sorokina ◽  
◽  
Yu. Yu. Stroilova ◽  
V. I. Muronets ◽  
Z. B. Shifrina ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Structural Characteristics ◽  
Short Review ◽  
External Factors ◽  
Amyloid Aggregation ◽  
Amyloid Proteins ◽  
Molecular Parameters ◽  
Amyloid Aggregates ◽  
Aggregation Processes

Among the compounds able to efficiently inhibit the amyloid aggregation of proteins and decompose the amyloid aggregates that cause neurodegenerative diseases, of particular interest are dendrimers, which represent individual macromolecules with the hypercrosslinked architectures and given molecular parameters. This short review outlines the peculiarities of the antiamyloid activity of dendrimers and discusses the effect of dendrimer structures and external factors on their antiamyloid properties. The potential of application of dendrimers in further investigations on the aggregation processes of amyloid proteins as the compounds that exhibit the remarkable antiamyloid activity is evaluated.

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