Enzyme Kinetics
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Complete list of Terms and Definitions for Enzyme Kinetics

Terms Definitions
hydrolases hydrolysis reactions
ΔS is + disorder inccreases
Cofactor ions needed for activity
Iodoacetamide group-specific irreversible inhibitor- covalently reacts with -SH groups
-dS/dt disappearance of substrate with time
Carriers Hemoglobin (O2), transferrin (Fe++), albumin (fatty acids)
Catalytic antibodies experimental proof for transition state stabilization by enzymes
Catalyst substance that speeds up chemical reaction w/out being changed after the reaction
Coenzymes specific heat-stable low MW organic molecules required for enzyme activity
Enzymes Resemble receptors and carriers but chemically modify ligand (catalysis). Most common type of protein (70% of known proteins)
Ki Affinity for any inhibitor.Ki = [i][E]/[ES]
ΔG ΔG°' + 2.303 RT log[P]/[S]The free energy change at non-standard conditions- determines whether or not a reaction is spontaneous in a cell
lyase cleave various bonds by means other than hydrolysis and oxidation
Reversible Inhibitor Effects on Lineweaver-Burk plot competitive: slope effectnoncompetitive: slope and y-intercept effectuncompetitive: y-intercept effect
V= d[P] /dt is also what? V= -d[S]/dt
Lineweaver-Burk Plots Mathematical conversion of M-M plot (hyperbolic) gives a linear plot. Intercept on the y-axis to determine Vmax and intercept on x-axis to determine Km
ΔG is + endergonic NOT spontaneous- must add energy for reaction to occur
Transition-State Stabilization - enzyme active site is complementary to the transition sate substrate- enzyme active site stabilizes those features of S which appear in transition state substrate - enzyme binds more tightly to the transition state substrate
Two Examples of Competitive Inhibitors succinate dehdrogenase and bisphosphoglycerate mutase
What units are Km expressed in? moles/liter or mg/ml
More than 50% of enzymes use metal (ions) cofactors. Zn++, Fe++, Fe+++, Mg++, Cu++, K+, Mn++ Coenzyme
Ki and competition Competition depends on affinities and concentrations of I and S. If Ki is less than Km, significant competition will occur. If [I] is close to Ki, significant inhibition will occur. Conc. of I must be half of conc. of E in order for Ki to occur.
Enzyme inhibition Inhibitors are usually analogs to the substrate and bind in the same place. Can be competitive or non-competitive (uncompetitive also exists). Ki only refers to reversible rxns. Covalent inhibition is irreversible.
Zygomen Precursor form of an enzyme that is only activated by proteolysis.
Potential Uses of Reversible Inhibitors 1) therapeutic agents2) experimental techniques3) Information about structure of enzyme and its active site
All E in the form of ES is what? Saturated
Substrate and Enzyme Concentrations Most current metabolites are normally found in levels appropriate to the enzyme that processes them. Enzymes have evolved to interact with substrates that best fit their function and have adjusted to be at the correct concentrations for this substrate.
Allosteric "Other form". Means an enzyme that is regulated by conformational change between active and inactive forms.
Competitive inhibitors Bind to same place as substrate and stop regular rxn from occurring. They can also add to another binding site, conformationally changing and de-activating enzyme. Competitive inhibitors do not actually change the affinity of enzyme for substrate, but do make Km ([S] for 50% bound) higher.
The Specificity Constant Kcat/Km- typically b/w .1 and 1- used to rank enzymes according to how well they utilize different substrates (catalytic efficiency)- upper limit of 10^8 to 10^9 M^-1sec^-1 (represents enzyme perfection)
1. Enzymes react with only very specific substrates to create specific products. 2. Enzymes do not change the Keq for a reaction.3. Enzymes increase the rate of the reaction by up to 10^17 (usually 10^5-10^9). Ubiquity of enzyme activity
A substrate with low dissociation rate (high affinity)for the enzyme would reach what? half-maximal velocity at lower [S]
Side Effects and Drug concentrations Drugs must be dosed specifically to target the enzyme they wish to inhibit. Too little will be insufficient to compete with substrate. Too much will cause likelihood of effecting/inhibiting other enzymes. This can cause "side effects". Most harmful consequences of clinical drugs are caused by these unintentional effects.
Association constant: Ka, Dissociation constant: KdSubstrate: KsInhibitor: KiUnspecified ligand: KL Always have units in molarity (micro or minimolar)
Key Features of Active Sites 1) take up relatively small part of total enzyme volume2) 3-D site formed by groups that come from different parts of the linear amino acid sequence3) has catalytic AA sidechains that bind to substrate and position the substrate4) is a cleft or pocket5) specificity of binding depends on the precisely defined arrangement of atoms in an active site
Km is sometimes associated with what? the strength of binding of the substrate for the enzyme.
1. Time. More time = more product as long as enzyme and substrate concentrations are available.2. Enzyme conc. More enzyme = more product.3. Substrate conc. If substrate conc is high, will cause product until amount of product is sufficient and rxn Binding constants
When saturation is at high [S] increasing the [S] doesn't do what? increase the rate of reaction any further
Dissociation constants and the 2-log rule Km and Ki signify the mid-point of the binding curve. The 2-log rule applies in this case as the affinities for substrate of enzymes are rarely variable. At a one log (or ten-fold) increase in concentration of the substrate, almost all of the enzyme will be loaded and active. At a ten-fold (or one log) decrease in the concentration of substrate, most enzymes will be empty. This is a hundred-fold total difference.
If [S]<Km v = Vmax[S]/Km
If [S]=Km v = Vmax/2
If [S]>Km v = Vmax
ΔS is - disorder decreases
+dP/dt appearance of product with time
Diisopropylfluorophosphate group specific irreversible inhibitor- covalently reacts with serine sidechains- inactivates chymotrypsin and acetlycholinesterase
Km A semi-dissociation constant. Affinity for the substrate. Defines the substrate concentration for 50% binding of the substrate or 50% action of the enzyme. Defined by the M-M equation.Km = [S]({Kcat[E]/Kobs}-1)Km = [S]({Vmax/v}-1)
oxidoreductases redox reactions with transfer of electrons
ΔH is - exothermic (gives off heat)
Change is entropy ΔS disorder or randomness
True or False Some enzymes approach the theoretical limit where close to every collision result in catalysis. True.
Metabolism Overall set of chemical reactions within a cell or organism.
V=Vmax[S]/[S]+Km is also what? Km=[S](Vmax-V)/V so when, 2V=Vmax, Km=[S]
Ks Affinity for the substrate. A dissociation constant. Ks = [S][E]/[ES]
Receptors Membrane bound (for hormones and other signals), immunoglobulins (for antigens), enzyme regulatory subunits (for regulatory metabolites)
Lineweaver-Burk plot 1/v vs. 1/[S]- y-intercept = 1/Vmax- slope = Km/Vmax- x-intercept = -1/Km
Cells far from the thermodynamic equilibrium are in what state? The steady state.
Non-competitive inhibitors are better In competitive inhibitors, substrate will build up due to non-processing, and will then out-compete inhibitor. Non-competitive is better.
We are interested in the affinity of enzyme to their substrate and the velocity of action (v). The affinity is quantified by Km, the Michaelis-Menten constant. Ki
Michaelis-Menten Equation V_o = V_max ([S])/([S]+ k_M ) -relates the rate of catalysis to [S] and [E] and also the rates of the individual steps
The larger the Kcat the more often what happens on the surface of the enzyme? catalytic events
Every chemical reaction in the human body uses at least one enzyme. Active site/catalytic site
Enzymes lower the free energy of reaction activation. This is the key to enhancing catalytic rates. Enzymes and pH
Types of Associations b/w Enzyme and Substrate H-bonds, hydrophobic interactions, salt bridges, and dipole-dipole interactions
Enzymes' affinity for their substrate The current "normal" concentrations of metabolites is controlled to be appropriate for the enzymes that use them. Also the enzymes have evolved to bind substrates that work well for their function and for the available concentration of the substrate.
Noncompetitive Inhibitors - binds to both E and ES- Vmax is decreased (when Ki=Ki')
Enzymes are effected by temp, but only at lower temps than body temp (37C). Higher temps increase non-catylized rxns, but at high temps, enzymes can be denatured. The lowest temp that works for enzymes in the body is the lowest temps the body can tolera Evaluation of Enzyme activity
What is Michaelis's constant? [S] is 1/2 the Vmax. The term is called the Km
Usually use induced fit to physically put substrate into position for faster rxn. Enzymes and free energy of activation
Units of Kcat are per what? What does Kcat measure? per seconds, that is (s^-1) measures number of substrate molecules turned over per enzyme per second. Values range from chymotypsin (0.1) to carbonic anhydrase (100,000).
Why these affinity constants are important For a drug to be effective, it must compete with the regular substrate. Therefore its affinity constant (Ki) must complete with the enzyme's affinity for the regular substrate (Km).