Substrate inhibition of lactose/proton symport in Escherichia coli

1980 ◽  
Vol 8 (6) ◽  
pp. 704-704 ◽  
Author(s):  
MALCOLM G. P. PAGE
Keyword(s):  
Escherichia Coli ◽  
Substrate Inhibition ◽  
Proton Symport

scholarly journals Alternative-substrate inhibition and the kinetic mechanism of the β-galactoside/proton symport of Escherichia coli

1982 ◽  
Vol 204 (3) ◽  
pp. 681-688 ◽  
Author(s):  
M G Page ◽  
Y H Jou
Keyword(s):  
Escherichia Coli ◽  
Substrate Concentration ◽  
Substrate Inhibition ◽  
Maximum Velocity ◽  
Kinetic Mechanism ◽  
Kinetic Constants ◽  
Alternative Substrate ◽  
Proton Binding ◽  
Alternative Substrates ◽  
Proton Symport

The effects of competing alternative substrates on the rate of uptake by galactoside/proton symport were investigated. These experiments produced a decrease in apparent maximum velocity with increased alternative-substrate concentration that cannot be accounted for by a simple ordered mechanism. This, together with non-linearities in the variation of the apparent kinetic constants with alternative-substrate concentration, can be accounted for by a random mechanism for galactoside and proton binding.

scholarly journals Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

2004 ◽  
Vol 70 (10) ◽  
pp. 6257-6263 ◽  
Author(s):  
Jyh-Shing Hsu ◽  
Yunn-Bor Yang ◽  
Chan-Hui Deng ◽  
Chia-Li Wei ◽  
Shwu-Huey Liaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Substrate Inhibition ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Penicillin G ◽  
Dna Shuffling ◽  
Homologous Genes ◽  
Family Shuffling

ABSTRACT Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k cat/Km ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

scholarly journals Stoicheiometry of lactose–proton symport across the plasma membrane of Escherichia coli

1973 ◽  
Vol 132 (3) ◽  
pp. 587-592 ◽  
Author(s):  
Ian C. West ◽  
Peter Mitchell
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Transport Processes ◽  
Proton Symport

The rate of [14C]lactose inflow into anaerobic, non-metabolizing, Escherichia coli has been correlated with the rate of the simultaneous effective inflow of H+. It is shown that the initial rates of the two transport processes are essentially the same, and it is concluded that the two processes are strictly coupled with a 1:1 stoicheiometry.

Inhibition of oxidative metabolism in Escherichia coli by d-camphor and restoration of oxidase activity by quinones

1975 ◽  
Vol 21 (9) ◽  
pp. 1357-1361 ◽  
Author(s):  
Maria A. Cardullo ◽  
James J. Gilroy
Keyword(s):  
Escherichia Coli ◽  
Vitamin K ◽  
Oxidative Metabolism ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Substrate Inhibition ◽  
Lower Surface ◽  
Coli Strain ◽  
Cell Permeability ◽  
Alumina Powder

Oxidative metabolism in whole cells of Escherichia coli strain 82/r was inhibited by d-camphor when glucose, pyruvate, or succinate was used as substrate. Inhibition was not due to lower surface tension in d-camphor-treated cell suspensions nor was it a function of cell permeability. Succinic, lactic, and NADH-oxidase activities were inhibited in alumina powder cell-free extracts (80 μg of protein/ml) by d-camphor (1100 μg/ml). NADH: and succinic: DCPIP oxidoreductase enzymes were unaffected by d-camphor. Menadione (vitamin K3) restored succinic, lactic, and NADH-oxidase activities in d-camphor-inhibited cell-free extracts. Concentrations of menadione used to restore succinic and NADH oxidase activities were not stimulatory in non-camphor-treated extracts. Succinic oxidase activity in d-camphor-inhibited cell-free extracts was also restored by ubiquinone (Q6) but not by vitamin K1. These results are interpreted to indicate that d-camphor may affect quinone function in E. coli.

scholarly journals The kinetics of the β-galactoside-proton symport of Escherichia coli

1981 ◽  
Vol 196 (3) ◽  
pp. 721-731 ◽  
Author(s):  
M G Page ◽  
I C West
Keyword(s):  
Escherichia Coli ◽  
Kinetic Constants ◽  
Protonmotive Force ◽  
Michaelis Constant ◽  
Transport Reaction ◽  
Proton Symport ◽  
Kinetics Of ◽  
External Ph

beta-Galactoside transport by Escherichia coli occurs with the concomitant uptake of a proton. The kinetics of beta-galactoside uptake at various values of external pH are interpreted in terms of a model in which both the galactoside and the proton are substrates of the transport reaction. The values of some of the kinetic constants for this two-substrate reaction were determined. The observed effects of the protonmotive force on the apparent Michaelis constant for galactoside can be explained in terms of the proton being a substrate of the transport reaction.

scholarly journals Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase

2008 ◽  
Vol 190 (7) ◽  
pp. 2556-2564 ◽  
Author(s):  
Cory G. Toyota ◽  
Catrine L. Berthold ◽  
Arnaud Gruez ◽  
Stefán Jónsson ◽  
Ylva Lindqvist ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Coenzyme A ◽  
Substrate Inhibition ◽  
Kinetic Behavior ◽  
Oxalobacter Formigenes ◽  
Structural Genomic ◽  
E Coli ◽  
Coa Transferase ◽  
Acidic Conditions

ABSTRACT The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

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