wk8-1.13-1.14:Enzymes and Enzyme kinetics

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wk8-1.13-1.14:Enzymes and Enzyme kinetics by Mind Map: wk8-1.13-1.14:Enzymes and Enzyme kinetics

1. Enzymes

1.1. Enzyme catalysis

1.1.1. increase the rate of chemical rxns

1.1.2. speed by a factor of 10^4 and greater

1.1.3. highly selective

1.1.3.1. only one particular rxn catalysed

1.1.3.2. activities can be regulated more easily

1.1.4. nearly all cellular rxns or processes are mediated by a protein(or RNA) catalysts called enzymes

1.1.4.1. biological systems dont use inorganic catalysts

1.1.4.2. all catalysis in cells carried out by organic catalysts= enzymes

1.1.5. 3 basic catalyst properties

1.1.5.1. increases the rate of rxn by lowering the Ea requirement,allowing thermodynamically feasible rxns to occur at a reasonable rate

1.1.5.1.1. no heat required

1.1.5.2. acts by forming transient,reversible complexes with substrate molecules, binding them in a way that aids their interaction and stabilizes the transition state

1.1.5.3. changes only the rate at which equilibrium is achieved

1.1.5.3.1. no effect on position of equilibrium

1.1.5.3.2. cannot make a rxn spontaneous

1.1.5.3.3. not thermodynamic wizards

1.1.5.4. apply to both inorganic and organic catalysts

1.2. most enzymes are proteins

1.2.1. primary structure

1.2.1.1. amino acid sequence

1.2.2. secondary structure

1.2.2.1. alpha helices

1.2.2.2. beta pleated sheets

1.2.3. tertiary structure

1.2.3.1. 3D organistion

1.2.4. quaternary structure

1.2.4.1. more than 1 polypeptide chain

1.2.4.2. e.g. antibodies

1.3. naming

1.3.1. end with suffix -ase

1.3.2. ECN (enzyme commision number )

1.3.2.1. e.g. EC 4.1.2.39

1.3.2.1.1. hydroxynitrilase

1.3.2.2. describes enzyme activity

1.3.3. 6 major groups

1.3.3.1. oxidoreductases

1.3.3.1.1. oxidation-reduction rxns

1.3.3.2. transferases

1.3.3.2.1. transfer of functional grps from one molecule to another

1.3.3.3. hydrolases

1.3.3.3.1. hydrolytic cleavage of one molecule into two molecules

1.3.3.4. lyases

1.3.3.4.1. removal of a group from, or addition of a grp to a molecule with rearrangement of electrons

1.3.3.5. isomerases

1.3.3.5.1. movement of functional grp within a molecule

1.3.3.6. ligases

1.3.3.6.1. joining of two molecules to form a single molecule

1.4. Activation Energy Barrier

1.4.1. activation energy (Ea)

1.4.1.1. minimum amount of energy that reactants must have b4 collisions between them can occur

1.4.1.2. minimum amount of energy required for the reactants to reach the transition state and give rise to products

1.4.2. rate of rxn

1.4.2.1. proportional to the fraction of molecules with energy =/> than Ea

1.4.3. transition state

1.4.3.1. an intermediate chemical stage that reactants need to reach

1.4.3.2. higher free energy than initial reactants

1.4.4. the only molecules that react are those with enough energy to exceed the Ea barrier

1.4.5. each rxn has a specific Ea

1.4.6. Metastable state

1.4.6.1. most biological rxns have high Eas

1.4.6.1.1. proportion of molecules with sufficient energy to react is very small

1.4.6.1.2. rates of uncatalyzed rxns in cells are very low

1.4.6.1.3. prevents most cellular rxns occurring in absence of a suitable catalyst

1.4.6.2. seemingly stable molecules that are potential reactants in thermodynamically favourable rxns

1.4.6.2.1. don't have sufficient energy to overcome Ea barrier

1.4.6.3. important because life is a system maintained in a steady state

1.4.6.3.1. never reaches equilibrium

1.4.7. some rxns=thermodynamically feasible but do not occur to an extent f

1.4.7.1. e.g. ATP hydrolysis to ADP

1.4.7.2. rxn is highly favourable and exergonic

1.4.7.2.1. especially in cell conditions

1.4.7.3. despite the highly favourable free energy change this reaction occurs only slowly on its own

1.4.7.3.1. ATP remains stable for a long time when dissolved in pure water

1.4.8. 3 ways to overcome the Ea barrier

1.4.8.1. increase the proportion of molecules with enough energy to react

1.4.8.1.1. increase average energy content of molecules

1.4.8.1.2. lower activation energy requirement

1.4.8.1.3. quantum tunneling

1.5. E-S complexes

1.5.1. enzymes are specific for their substrate

1.5.1.1. binds to complementary active site on the enzyme molecule

1.5.1.1.1. every enzyme has at least 1 active site

1.5.1.1.2. not altered or/consumed by rxn

1.5.2. Lock and key theory

1.5.2.1. 1594 Fisher

1.5.2.2. key=substrate

1.5.2.3. lock =enzyme

1.5.3. induced fit model

1.5.3.1. distorted both the substrate and the enzyme

1.5.3.1.1. undergo conformational change

1.5.3.1.2. substrate becomes susceptible to catalytic attack

1.6. co-factors and co-enzymes

1.6.1. cofactors

1.6.1.1. additional non-protein molecules needed for catalytic activity

1.6.1.2. prosthetic grps

1.6.1.3. inorganic

1.6.2. coenzymes

1.6.2.1. small organic molecules

1.6.2.1.1. derivatives of vitamins

1.6.2.1.2. normally found in enzyme active sites

1.7. Factors that affect enzyme activity

1.7.1. pH

1.7.1.1. most enzymes in the body have a pH 3-4

1.7.1.2. optimal pH depends on location of enzyme in the body

1.7.1.2.1. e.g. trypsin

1.7.2. temperature

1.7.2.1. optimal temp

1.7.2.1.1. different for different enzymes

1.7.2.1.2. e.g. typical human enzyme has optimum temp at 37C

1.7.2.1.3. below and above optimal temp=denaturation

1.8. Enzyme regulation

1.8.1. the cell regulates the catalytic activity of enzymes

1.8.2. enzymes generate a complex web of metabolic pathways

1.8.2.1. series of chemical rxns

1.8.2.2. product of one rxn =substrate of another

1.8.3. Control Mechanisms

1.8.3.1. feedback inhibition

1.8.3.1.1. end product feeds back to inhibit the rxn

1.8.3.1.2. 2 types

1.8.3.1.3. multiple feedback inhibition

1.8.3.2. Allosteric regulation

1.8.3.2.1. most important control mechanism

1.8.3.2.2. allostery

1.8.3.2.3. regulatory molecule has a diff. shape

1.8.3.2.4. allosteric enzymes

1.8.3.2.5. allosteric effectors

2. Enzyme kinetics

2.1. enzyme inhibition

2.1.1. control mechanism in cells

2.1.2. inhibiting specific enzymes(drugs and poisons)

2.1.3. effective tools

2.1.3.1. rxn mechanisms + for treatment of diseases

2.1.4. 2 types of enzyme inhibitors

2.1.4.1. irreversible

2.1.4.1.1. binds covalently

2.1.4.1.2. causes permanent loss of catalytic activity

2.1.4.1.3. toxic to cells

2.1.4.1.4. therapeutic agents

2.1.4.1.5. penicllin

2.1.4.2. reversible

2.1.4.2.1. binds in a non-covalent dissociable way

2.1.4.2.2. competitive

2.1.4.2.3. non competitive

2.2. describes

2.2.1. quantitiave aspects of enzyme catalysis

2.2.2. the rate of substrate conversion

2.2.2.1. initial rxn rates

2.2.2.1.1. when the substrate conc. has not yet decreased enough to effect the rxn rate

2.3. Michaelis-Menten kinetics

2.3.1. initial velocity (v)

2.3.1.1. changes depend on the substrate conc.

2.3.1.2. tends towards Vmax

2.3.1.3. rate of change in product conc. per unit time

2.3.1.3.1. mM/min

2.3.1.4. rxn velocities are experimentally measured in a constant assay volume of 1mL and are reported as micromoles of product per minute

2.3.2. substrate conc. (s)

2.3.2.1. if s=very large

2.3.2.1.1. maximum velocity(Vmax) is reached

2.3.2.2. tends towards infinity

2.3.3. Km

2.3.3.1. corresponds to substrate conc.

2.3.3.2. v=initial rxn velocity

2.3.3.3. [S]=initial substrate conc.

2.3.3.4. Vmax=maximum velocity

2.3.3.4.1. michaelis's constant

2.3.3.5. Km=concentration of substrate that gives exactly half the maximum velocity

2.3.3.6. 3 cases

2.3.3.6.1. very low substrate conc.

2.3.3.6.2. very high substrate conc

2.3.3.6.3. [S]=Km

2.3.4. saturation

2.3.4.1. the inability of higher saturation

2.3.4.2. all active sites are occupied and enzymes working at max velocity

2.3.4.3. Ef=free form of enzyme

2.3.5. Importance of Km and Vmax

2.3.5.1. Km

2.3.5.1.1. useful because it allows us to estimate where along the Michaelis-Menten plot an enzyme is functioning in a cell

2.3.5.1.2. can estimate at what fraction of the maximum velocity the enzyme catalyzed rxn is likely to be proceeding in the cell

2.3.5.1.3. the lower the Km value for a given enzyme and substrate

2.3.5.1.4. regulatory molecules can alter Km

2.3.5.2. Vmax

2.3.5.2.1. provides a measure of the potential maximum rate of the rxn

2.3.5.2.2. used to determine another parameter

2.3.5.2.3. Kcat

2.3.5.3. by knowing Km,Vmax,substrate conc

2.3.5.3.1. can estimate the likely rate of the rxn under cellular conditions

2.3.6. Double reciprocal plot

2.3.6.1. the michaelis-menten plot of v versus [S]

2.3.6.1.1. shows that velocity is dependent on substrate concs

2.3.6.1.2. not a useful tool for quantitative determination of Km and Vmax

2.3.6.2. Hans Lineweaver and Dean Burk 1934

2.3.6.2.1. converted the hyperbolic relationship of the MM plot into a linear function

2.3.6.2.2. double reciprocal plot=linear

2.3.6.3. Vmax can be determined directly from the reciprocal of the y-intercept and Km from the negative reciprocal of the x-intercept

2.3.6.3.1. slope can be used to check both values

2.3.6.4. useful experimentally because Vmax and Km can be determined without complication of hyperbolic shape

2.3.6.5. long extrapolation is often necessary to determine Km

2.3.6.5.1. may introduce uncertainty