BCH210H1F Enzymes notes.pdf

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Stavroula Andreopoulos

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BCH210 Enzymes  Proteins that catalyze chemical reactions 3 7  Catalytic power 10 - 10 times faster than no enzyme  Speeds approach to equilibrium but does not affect equilibrium  Lowers energy barrier, reach transition state faster 1. Enzyme Classes a. Oxidoreductase: oxidation-reduction reactions eg. Lactate Dehydrogenase oxidize L-Lactate to Pyruvate and reduce coenzyme NAD+ to NADH  Oxidases, dehydrogenases, reductases  One substrate is oxidized (lose electron) while the other is reduced (gain electron)  Involves coenzymes NAD+/NADH, FAD+/FADH  reversible b. Transferases: group transfer reactions eg. Alanine Transaminase, Carnitine acyltransferase  Kinases carry out phosphorylation reactions -> irreversible, requires ATP c. Hydrolases: catalyze hydrolysis, use H2O as substrate eg. Pyrophosphatase uses water to break pyrophosphate, hydrolysis of anhydride bond (very high energy linkage), gives 2 phosphates.  Protease, lipase, nuclease, esterase d. Lyases: create a double bond or addition to a double bond eg. Pyruvate Decarboxylase removes carboxyl group of Pyruvate and gives Acetaldehyde and CO2  Synthases catalyze addition reactions, breaks double bond  Reversible e. Isomerases: catalyze isomerization  Move around functional groups  Maintain chemical formula  Reversible f. Ligases: catalyze joining of two molecules eg. Addition of NH4+ with L-Glutamate to produce L-Glutamine  Requires energy (ATP, NADH, FADH) 2. Enzyme Specificity  High degree of substrate specificity Eg. Urease (bacterial enzyme) is specific for Urea, hydrolyse Urea into 2NH3 and CO2  Specificity for a functional group Eg. Alcohol dehydrogenase: act on any alcohol group, oxidoreductase  Broader specificity o Hexokinase: catalyze addition of phosphate to hexoses sugars  Uses ATP to drive production of sugar-phosphate ester  Glucose to glucose 6-phosphate o Proteases: hydrolyze peptide bonds and some also hydrolyze esters o Specific proteases  Bacterial protease subtilisin  Specificity for amino acids: trypsin cleaves K and R on carboxyl side 3. Enzyme Active Site  Enzyme specificity and efficiency are controlled by active site  AS: specialized region in enzyme for S binding and catalysis  AS formed during protein folding, has distinct 3D structure  When S binds to AS, helps promote formation of transition state – speeds up reaction  AS has unique microenvironment  1-6 key residues, 2-3 carry out catalysis  Reactants brought together, form mostly H-bonds  AS is a small % of total volume of enzyme e.g. Lysozyme: enzyme in tears, saliva - attacks bacterial cell wall polysaccharide - serves as antibiotic - at pH5, Asp52 is deprotonated, Glu 35 is protonated – acid base catalysis - six binding pockets A-F, binds GlcNAc and MurNAc alternate - cleaves 1-4 glycosidic linkage of D and E residues  Lock and Key model: E and S are complementary shapes that fit together  Induced-fit model: enzyme changes conformation upon binding of substrate o E.g. Hexokinase uses induced-fit to prevent water from coming into active site and interfere with reaction 4. Enzyme Regulation 1) Substrate availability and supply: cannotmake product unless substrate is available. In certain pathologies, a substrate becomes available. o E.g. injury causes activation of phospholipase A2 which degrade membrane phospholipid  wounding response  generate Arachidonic Acid (polyunsaturated FA, omega-6, 20C:4), which is converted to Eicosanoids by Cyclooxygenase (COX) and causes inflammation responses. 2) Covalent Modification: enzyme is changed chemically resulting in changes in conformation and activity o E.g. OH group in Ser, Thr, Tyr phosphorylated by protein kinase, phosphatase can restore the OH group  Pyruvate dehydrogenase is inactive after being phosphorylated, active after hydrolyse by pyruvate dehydrogenase phosphatase  Activation of phosphorylase b to phosphorylase a to breakdown glycogen to glucose-1-P  Depending on enzyme, phosphorylation may give activation or inactivation  Reversible reaction 3) Allosteric Control: reversible binding a modulator at second site on enzyme changes enzyme shape and activity o Tertiary structure: modulator binds allosteric site o Quaternary structure: modulator binds to allosteric site on regulatory subunit in multi-subunit proteins o A. Feedback Inhibition:  Chain of enzymes convert Thr to Ile  Ile inhibits first enzyme in pathway, negative feedback  Feedback at E1 to save resources, reduce intermediates o B. Energy Status Control:  PFK-1 involves in production of ATP in glycolysis, activated by ADP (modulator)  When ATP level is low, ADP level is high, PFK-1 is allosterically activated to produce more ATP o C. Lose of Allosteric Control:  Gout  Lack of feedback inhibition by purine nucleotides on PRS, accumulation of URATE 4) Zymogens, Proenzymes: inactive enzymeprecursors o Inactive Trypsinogen is cleaved by Enteropeptidase off 6 AA at N-terminus to produce active Trypsin in stomach and intestine o Trypsin positively feedback to the conversion of Trypsinogen to Trypsin – self sufficient o Trypsin activates Chymotrypsin and Elastase 5. Enzyme Kinetics  Keq = [P]/[S]  [ ] = concentration at equilibrium (moles/L)  Vo is enzyme velocity (rate of product formation)  Vo is proportional to [substrate], hence Vo = k[S], k=rate constant  In [P] against time graph, Vo departs from linearity o [substrate]↓ o Inhibition by product o Enzyme instability o If ↑[substrate], Vo↑  In Vo against [substrate] graph, as [S]↑, Vo → Vmax (limiting value) o Hyperbolic plot  Michaelis-Menten Kinetics o Rate of P formation: v=k2[ES] o Rate of ES formation: k1[E][S] o Rate of ES breakdown: (k-1+k2)[ES] o Steady state: k1[E][S]=(k-1+k2)[ES] formation=breakdown  Km: kinetic activator constant o Constant derived from rate constants o Half Vmax o Estimate of affinity of substrate to enzyme o Higher Km indicates less affinity o Physiological consequence of Km  Ethanol is broken down by alcohol dehydrogenase to acetaldehyde, which is then converted to acetate by acetaldehyde dehydrogenase. The second reaction has a high Km, hence acetaldehyde accumulate in blood after too much alcohol consumption. Acetaldehyde dehydrogenase exists in both mitochondrion and cytoplasmic, and has lower Km in mitochondrion. The diffusion of acetaldehyde from cytoplasmic to mitochondrion takes time; hence time is needed for alcoholic effects to be elevated
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