Enzyme kinetics are the studies of the biophysical properties of enzymes and their substrates.

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Convert flux value in nmol/min/mg protein to nmol/min/cell?

This is a question about unit conversion. I found an experimental measurement of the saturation uptake of glucose in yeast: $$V_\textrm{max} \approx 650 \textrm{nmol}/\textrm{min}/\textrm{mg ...
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49 views

Do we actually know the molecular dynamics of any enzyme?

That is right, is there a limitation, say Heisenberg's uncertainty principle or something that limits our understanding of machinery of enzymes at atomic level? Can we know how do they actually work? ...
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Is it possible to measure in vivo enzyme kinetics?

I was wondering is there way in which we can measure enzyme kinetics in vivo specifically for enzymes acting on lipid substrates (where substrate is restricted to 2D membrane as oppose to freely ...
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29 views

kinetics question [closed]

An enzyme has a $V_{max}$ of 50 $\mu$mol product formed $(min * \text{mg protein})^{-1}$ and a $K_m$ of 10 $\mu$M for the substrate. When a reaction mixture contains the enzyme and a 5$\mu$M ...
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279 views

Does pH affect Michaelis constant?

I have been trying to confirm the Km of a substrate (which is 34 +/- 4 mM). This value was obtained in 50 mM MOPS, pH 6.3. I conducted my kinetics assay in a buffer of pH 7 and obtained a Km value in ...
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37 views

Is NADPH unstable in UV light?

I am working on an enzyme activity assay using NADPH as a cofactor. The MSDS for NADPH Tetrasodium Salt, tells to store it in a place away from heat and light. Does this include UV light or just ...
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399 views

The effect on the efficacy and potency of a non-competetive antagonist binding to the active site of the receptor (dose-response curve)

According to the book "Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy" by Golan et al, non-competetive antagonists can bind to both the allosteric site and the active site. I ...
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68 views

DNA length and annealing kinetics

I have a mixture plasmids and undesired short linear fragments that share the same sequences. During denaturation and annealing, I would like the plasmids to 'find each other' before annealing to the ...
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220 views

Is there a formula for determining the % of ligand bound to a given receptor in a mixture with multiple ligands and receptors?

When a ligand and receptor interact with a given kd, one can easily determine % of ligand bound to the receptor when the kd, total concentration of ligand, and total concentration of receptor are ...
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113 views

Enzymatic error rate

I am aware that each enzyme generate a certain amount of misproducts. This is well documented, for example, for the DNA polymerase. I am interested in enzyme involved in biochemical processes, so for ...
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851 views

Making the sense of enzyme Km comparisons

I have encountered comparisons of the Michaelis-menton constant (Km) a few times. Generally speaking if Km of an enzyme is higher, then it's affinity to its substrate is lower. How does this make ...
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93 views

Mode of Enzymatic Inhibition via R-Allele

The photo above shows the effects of the R allele of the pea shape gene on the synthesis of an enzyme that converts unbranched starch into branched starch. The r allele of this gene determines an ...
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Kinetic binding / unbinding constants of protein + mRNA ⇌ mRNP

I wish to find an order of magnitude for the binding/unbinding constants of mRNA and RNA-binding proteins. Suppose molecules A and B reversibly associate to give the complex C. Assuming the diffusion ...
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620 views

Why would an Eadie-Hofstee Plot be non-linear? [closed]

Besides cooperativity between multiple active sites on an enzyme, what are the other reasons for the Eadie-Hofstee plot to be non-linear?
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150 views

Enzyme Assay - pectinase

During assaying an enzyme at high temperature, the substrate (Pectin) is degraded by the high temperature rather than by enzyme, so, how can I minimize degradation of the substrate by the temperature? ...
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1answer
93 views

can a bacterial protease inhibitor cocktail be used for Western Blotting involving HUVEC cells and HT-29 adenocarcinoma cells?

Dear fellow biochemists, I need some advice on Western Blotting, more specifically the use of certain protease inhibitors with the RIPA cell lysis buffer and protease inhibitor cocktail. A Millipore ...
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1answer
100 views

Pectinase Enzyme Assay

I am working on pectinase enzyme assay. I incubated 900 ul of substrate for 10 minutes in the water bath, followed by adding 2ml of DNSA reagent, then 100ul of enzyme extract added finally i read the ...
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1answer
939 views

DpnI over-digestion

We have a long protocol that we are optimizing that includes DpnI digestion of a PCR product (to remove any of the template DNA if it's methylated, and while we're not certain in the blind tests, ...
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1answer
115 views

What does “thermodynamic equilibrium” mean for an enzyme-substrate complex?

From Fersht, Enzyme Structure and Mechanism p. 87: The Michaelis-Menten mechanism assumes that the enzyme-substrate complex is in thermodynamic equilibrium with free enzyme and substrate. In ...
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What's the difference between Ki and IC50?

I have just read this post at researchgate: "How to calculate experimental binding free energy from the IC50 value" I am not be very sure about what the difference is between Ki and IC50. Could ...
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75 views

Enzyme kinetics [closed]

I can't understand how to study enzyme kinetics. Say I have a lipase and want to study the kinetics of this lipase using a fluorogenic substrate, how would I do this? From what I understand I would ...
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42 views

Activity of glucokinase

From Solomon et al, 2013 ACC Synthetic biology and from this video : Here, there are 2 competing reactions for glucose - one with glk as enzyme and other with gdh as enzyme. In the graph, y axis ...
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551 views

How to calculate or know by experiment the entropy of enzymes or protein?

How do you calculate or experimentally determine the entropy of enzymes or protein? In particular, I am interested in Boltzmann and conformational entropy, and Gibbs free energy. Any references are ...
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187 views

Are there any methods to quantify H2O2 (hydrogen peroxide) which don't rely on horseradish peroxidase?

I can't use HRP since my substrates interfere with it and I need a real-time method because I want to measure kinetic parameters of some oxidases.
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741 views

Why are enzyme-catalysed reactions slower at lower substrate concentration?

Suppose I'm using 200 nmoles of enzyme and 2 mmoles of substrate. The enzyme should be saturated but if I use 50 mmoles of substrate, the reaction will be faster. Why? I just can't get it! Even at ...
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100 views

Modeling yeast biochemical pathway using enzyme kinetics

I need enzyme concentration and metabolites concentration values to en-corporate these values in my model and do some simulation. I searched through some database yeast metabolome database ...
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764 views

What is the use of futile reaction cycling such as Fruc-P to Fruc-BP?

One rate-limiting step of glycolysis is the conversion of Fructose-Phosphate (Fruc-P) to Fructose-Bisphosphate (Fruc-BP), catalysed by Phosphofructokinase (PFK). The reaction involves hydrolysing one ...