Biochem 2

najudexi's version from 2016-09-25 06:26


Chapter 8 Enzymes as Catalysts
• Enzyme-cataylzed reaction (E+SES, ES EP, EPE+P) enzymes binds substrate bringing into correct orientation, break bonds, release products and returns to original state (Catalytic Power 106 to 1014)
• The active site has functional groups directly participating in reaction, conformational changes will occur with a transition state complex (unstable high energy)
o Lock and Key model can use hydrophobic interactions, electrostatic interaction, H bonds (coming from different parts of AA sequence) as a “perfect fit”
o Induced Fit occurs when substrate binds causing conformational change (thus increasing number reactions), usually repositioning functional groups
• Transition State complex encompasses highest point with greatest energy required, energy barrier has to be overcome and substrates have to be activated by the activation energy for the reaction to proceed (enzymes will decrease activation E)
• Chymotrypsin is a digestive enzyme in intestine for specific peptide bonds, member of serine protease superfamily (use active site of serine to form covalent intermediate), functional groups activate the –OH group of serine to attack carbonyl carbon, forming an oxyanion covalent intermediate (and thus stabilizing), while destabilizing the leaving group
o Without chymotrypsin present water’s –OH will attack carbonyl C, forming an unstable oxyanion tetrahedral in the TS (O has negative charge), process being slow due to the few number of hydroxyl groups with enough energy to attack correctly
o Step 1 (substrate binding) – specifically hydrolyzes peptide bond on carbonyl side of Phe, Tyr, Trp (in denatured protein), hydrophobic pocket for hydrophobic AA in bond allowing proximity and orientation
o Step 2 (histidine activates serine) – Asp and His (acid base catalysis for His) make Ser’s –OH more nucleophilic (attracted to + charge), attacking carbonyl C of scissile bond, Asp-His-Ser are catalytic triad engaging in cooperative interactions
o Step 3 (oxyanion tetrahedral TS stabilized by H bonds) – amino group (NH) from backbone stabilizes and lowers TS energy level
o Step 4 (cleavage of peptide bond) – full covalent between –OH of Ser and carbonyl C, peptide bond cleaved in covalent catalysis by the negatively stabilized O anion reforming double bond and cleaving C-N bond
o Step 5 (covalent acyl-enzyme intermediate) – the amino group that formed is now destabilized with His present (destabilization of developing products aka leaving group)
o Step 6 water molecule attacks carbonyl carbon Step 7 forms second oxyanion tetrahedral (also stabilized by H bonds) Step 8 acid catalysis breaks the acyl-enzyme covalent bond (negatively stabilized O anion reforms double bond, cleaves C-O) Step 9 product is free to dissociate
• Functional groups in catalysis – proximity and orientation (in all enzymes), all enzymes stabilize TS by electrostatic interactions, not all form covalent intermediates, cofactors can be used (coenzymes, metal ions, metalloceoenzymes), and in AA side chains covalent catalysis (Ser/Cys/Lys/His ), acid base catalysis (His), nucleophilic polar
o Coenzymes are nonprotein organic molecules (usually from vitamins)
♣ Activation-Transfer Coenzymes form covalent bond with substrate (activates for transfer, separate portion of coenzyme binds the protein, little activity and specificity in absence of the enzyme
• TPP (thiamine pyrophosphate) derived from Vitamin Thiamine (B1) and pyrophosphate, charged O in pyrophosphate chelated by Mg2+ binds to enzyme, functional group interacting with active site is reactive C (with dissociable proton in thiamine portion) forming covalent bond with substrate keto cleaving adjacent C-C (pyruvate), decarboxylation, resonance also makes stable
o Thiamine containing enzymes cleave at different sites
• Coenzyme A from Vitamin Panthothenate (B5), adenosine 3’5’-biphosphate binds tightly but reversibly to enzyme, nucleophilic sulfhydryl –SH attacks carbonyls forming acyl thioesters and covalent bond intermediate
• Biotin (B7, biocytin complex) has no phosphate group, Lys in carboxylase binds enzyme covalently, functional group nitrogen activates and transfers carbon dioxide covalently, does carboxylations
• Pyridoxal Phosphate (PLP) from Vitamin pyridoxine (B6) is in transamination reactions, removing nitrogen from AAs, degrading/synthesizing AAs (AAs!), functional group aldehyde (unknown interaction with enzyme)
♣ Oxidation-Reduction Coenzymes (involved with oxidoreductases), NAD+ and FAD can transfer electrons with H/dihydride, others work with metals to transfer a single electron to O, Vitamin E/C (ascorbic acid) act as antioxidants (protect against free radical), do not form covalent bonds
• NAD+ gains electrons from lactate dehydrogenase, made from Vitamin niacin and ATP, ADP portion binds enzyme, functional group is C in nicotinamide ring (accepting hydride)
o Lactate dehydrogenase example will create keto from alcohol, NADH dissociates
• Metal Ions have positive charge act as electrophiles (attract electrons), assist in binding/stabilizing, accept/donate electrons in redox, some bind multiple ligands (can be part of binding substrates/coenzymes/enzymes)
o Zinc and Alcohol Dehydrogenase, bind anionic substrates or intermediates, active site Ser pulls proton off ethanol hydroxyl O has negative charge, Zinc stabilizes allows transfer of hydride to NAD+
• Noncatalytic roles include cofactors serving structural roles by binding different regions to help form tertiary structure, can serve as substrates cleaved during reaction
• Optimal pH and Temperature
o Most enzymes work best at physiological pH range and 37 degrees C, curve shape depends on protonation state of active site residues, exception is pepsin ideal pH 1.6
• Mechanism Based Inhibitors (mimic/participate in intermediate step) such as covalent inhibitors form tight bonds with functional groups, DFP (prototype nerve gas)/Sarin/insecticides (malathion/palathion) all prevent enzyme from degrading NT ACh
o Aspirin covalently acetylates Ser in prostaglandin endoperoxide synthase
o TS Analogs/Compounds resembling Intermediate stages reaction, so potent because bind enzyme stronger than substrates (effective drugs do this at intermediate stages)
♣ Penicillin TS analog tightly binds glycopeptidyl transferase (required for cell wall synthesis), the penicillin beta-lactum resembling TS of natural reaction, suicide inhibitors undergo partial reaction to form irreversible inhibitors in active site
♣ Allopurinol (used to treat gout) causes xanthine oxidase to commit suicide by converting drug to TS analog
• Xanthine oxidase normally oxidizes hypoxanthine to xanthine and then xanthine to urates
• The enzyme contains Mo-S complex that binds substrates and transfers electrons required for oxidation, oxygen donated from water, can work as either an oxidase/dehydrogenase
• Allopurinol reacts with xanthine oxidase to produce Alloxanthine (oxypurinol), the slight difference in the orientation of the CH=N group of hypoxanthine versus Allopurinol, thus inactivating the enzyme
• Heavy Metals toxicity caused by binding of mercury Hg, lead Pb, aluminum Al, iron Fe to functional group of enzyme
o Hg binds many sulfhydryl active site groups, Pb replaces normal metal in enzyme causing developmental/neurological toxicity (involving Ca2+ in CNS, calmodulin, PKC)
• Oxidoreductase – Redox, Transferase – transfer functional groups, Hydrolases – break ester bonds, Lysase – break other than hydrolysis/oxidation, Isomerase – isomeric change, Ligase – covalent bonding


Chapter 9 Regulation of Enzymes
• Most pathways are usually feedback regulated, storage/toxic disposal pathways usually feedforward (reflecting availability of precursor), regulatory enzymes usually tissue specific, compartmentation collection of enzymes with common function in particular organelle/cell site
• Michaelis-Menten Equation
o E + S ES P (k1 from E+S to ES, k2 from ES to E+S, k3 is ES to P
o Vi = (Vmax x (S))/(Km + (S)), Vi = initial velocity, Vmax = maximal velocity achieved at infinite (S), Km = (S) required to reach half Vmax, Vi is proportionate to concentration enzyme-substrate complexes (ES), as (S) increases (ES) increases and rxn rate increases
♣ Km = (k2 + k3)/k1, Vmax = k3 (Etotal)
o Infinitely high S all E is bound to S, rate at Vmax, approaching limit of Vmax called saturation kinetics, the higher the Km the higher the (S) needed to reach half Vmax
• Hexokinase isozymes Km for Glucose
o Hexokinase I (RBC) Km .05 mM, Glucokinase (liver, pancreas) Km 5.5 mM (after fasting glucose about 5mM, after meal promotes storage)
• Reaction rate directly proportional to (E), Vmax often expressed as product per min/mg of enzyme not dependent on concentration, when E has more than 1 S apparent value of Km, many multisubstrate E (like glucokinase) do not fit M-M model but Km still used
• Reversible Competitive Inhibition competes with S for binding, usually structural analog, increasing substrate can overcome, competitive inhibitors increase apparent Km, no effect on Vmax (have to say “with respect to”)
o Noncompetitive Inhibition (reversible) does not compete with S for binding is noncompetitive with respect to particular S, an increase in S will not prevent noncompetitive inhibitor from binding the active site of another S, thus lowering Vmax
• Regulation via Conformational changes
o Allosteric activation/inhibition, phosphorylation or other covalent modification, protein-protein interactions, proteolytic cleavage
♣ Allosteric enzymes bind allosteric site causing a conformation change, usually have multiple (2+) interacting subunits active/inactive conformations, S binding to one facilitates another, S has difficult binding to T or tense conformation (low affinity) to change it to R or relaxed
♣ Allosteric effectors (activators/inhibitors) bind outside active site causing conformational change to catalytic site, activators bind more tightly to R (increase amount of active state E), inhibitors to T (increase S or activator to overcome)
• More effective than competitive/noncompetitive inhibitors
♣ Phosphorylation involves many E’s that are regulated by protein kinase/phosphatase, Ser/Thr/Tyr protein kinases use ATP to phosphorylate hydroxyl groups, causes conformational change (can increase/decrease activity)
• Muscle Glycogen Phosphorylase has rate limiting E for glycogen degradation from glycogen to glucose 1-P, the allosteric activator being AMP, creates active muscle glycogen phosphorylase
• Glycogen phosphorylase kinase links activation muscle glycogen phosphorylase to adrenaline by ATP breakdwon, phosphate removed by protein phosphatase 1
• Protein Kinase A is Ser/Thr protein kinase that phosphorylates many enzymes (including glycogen phosphorylase kinase), adrenaline increases cAMP binds regulatory subunit PKA (phosphorylation cascade) being adrenaline cAMP PKA phosphorylase kinase glycogen phosphorylase
• Protein-protein interactions include Calcium-calmodulin family of modulator proteins, conformation change/steric hindrance at active site (allosteric effectors), dissociable modulator protein
o Nerve impulse-Ca2+ release-calmodulin subunit of muscle glycogen phosphorylase kinase-conformational change-phosphorylates glycogen phosphorylase-ATP-contraction
• G Proteins are masters of regulation via protein association, small single unit binds and hydrolyzes GTP (active when bound to GTP), G proteins regulated by GTPase activating proteins (GAPs), guanine nucleotide exchange factor (GEFs), and GDP dissociation inhibitor (GDIs)
• Ras superfamily of small G proteins (Ras, Rho, Arf, Rab, Ran) for major regulation of growth/morphogenesis/motility of cell/axonal guidance/cytokinesis/trafficking, attached to membrane by myristoyl or farnesyl, regulates cell proliferation by GFs, SOS (GEF) binds Ras and promotes GDP dissociation, activated Ras binds protein kinase Raf (activation of transcription of genes)
• Proteolytic Cleavage with proenzymes being precursor proteins that must undergo this cleavage to become functional, zymogens are precursor proteins of proteases, change name with suffix “ogen” or prefix with “pro”
• Regulation of enzyme synthesis by induction/repression gene transcription, stabilization of mRNA (overall slow in humans), regulated protein degradation within characteristic half-life in lysosomes, proteasomes and caspases are specialized system for selective degradation
• Regulation of Metabolic Pathways
o Series of sequential reactions (product of one reaction is substrate of next), regulation occurs at rate-limiting step (slow, nonreversible first committed step)
o Feed-forward regulation can occur in disposal of toxic compounds (increase S to E of high Km, allosteric activation, substrate-related induction gene transcription, increase hormone), tissue-specific isozymes may have different regulation
o Counter-regulation of opposing pathways, and substrate channeling through compartmentation into complexes/organelles
o Feedback regulation uses pathway end product to control its own synthesis rate, usually involving allosteric regulation of rate-limiting enzyme by end product


Chapter 10 Cell Biology and Biochemistry
• Plasma membrane – selective restriction, proteins/lipids lateral movement, integral span membrane peripheral do not, glycocalyx carbohydrates from glycoproteins/lipids
o Mostly phospholipids, glycerol lipid phophatidylcholine, sphingolipid sphingomyelin both in outer, phosphatidylinositol in inner (signal transduction/NTs)
♣ Cholesterol between increasing fluidity, overall membrane is dynamic
o Transmembrane proteins are channels/receptors/transporters/structural, peripheral are bound by weak electrostatic interactions (spectrin example of mechanical support)
o GPI anchor covalently attached lipid anchors proteins to external surface
o Glycocalyx 2-10% membrane weight, protects against digestion, restrict uptake hydrophobic compounds, glycoproteins branched oligosaccharides (15 residues) via N/O-glycosidic bonds to Asn/Ser, glycolipids serve as recognition molecules
• Peroxisomes involved in oxidative reaction using molecular oxygen, toxic Hydrogen Peroxide produced in organelle is degraded in peroxisome, oxidizes long FA to shorter chains, converts cholesterol to bile salts (no DNA, replicates by cell division)