Enzyme

  • Define enzyme
    • en=in; zyme=yeast
    • biological catalyst increase rate of reaction by 1 mil-fold, typically about 1 thousand-fold
    • most enzymes are proteins
    • PM enzyme→regulate catalysis within cells in response to EC signals
    • circulatory enzymes→regulate blood clot
    • IC enzyme→catalyze reactions of metabolic pathways
  • Define substrate→molecule that binds to an enzyme to yield a product
  • Nomenclature
    • substrate→lactase catalyze lactose into glucose + galactose
    • action→DNase catalyze DNA into dNMP
  • Enzyme class 1 main class.1 subclass.1 sub-sub.1 serial no of sub-sub
    • oxireductase
      • +/- H
      • catalyze oxidation-reduction
      • 22 subclass
      • oxidation of alcohol donor + acceptor NAD+ to aldehyde + NADH→oxidation
      • active site→zinc, His-51, 67, Cys-174, 46, Ser-48, IIe-269, Val-292, Ala-317, Phe-319
    • transferase
      • transfer functional group→amino, acyl, P, glycosyl
      • 9 subclass
      • ALT→glutamic acid + pyruvic acid to a-ketoglutaric acid + alanine
    • hydrolase
      • nearly all proteins on outer surface→glycoprotein
      • hydrolysis of chemical bond
      • breakdown substrate by + H20
      • 13 subclass
      • serine protease cleave peptide bond
    • lyase
      • remove H2O NH3 CO2
      • cleave C-C, C-O, C-N, C-S, C-H, P-O bond
      • 7 subclass
      • pyruvate decarboxylase→pyruvate + H to acetaldehyde + CO2
      • depends on cofactors TPP and Mg
      • 4 active site, each has 20 aa, Glu-477 stability of TPP ring, 51 cofactor binding
    • isomerase
      • L to D isomerization
      • heterogenous group of enzymes catalyze isomerization of cis-trans and aldose-ketose interconversion
      • 6 subclass
      • D-glucose to trans enediol to D-fructose to cis enediol to D-mannose
    • ligase
      • 2 chemical group joined with high-energy phosphate bond of ATP is broken→ligate = bind
      • 9 subclass
  • Zymogen/proenzyme
    • inactive
    • trypsin→chymotrypsinogen to chymotrypsin
    • enteropeptidase→trypsinogen to trypsin
    • prothrombinase→prothrombin to thrombin
  • Simple enzyme→proteins + relatively small organic molecule
  • Isoenzyme
    • catalyze same reaction
    • differ structure (aa seq), Km, heat stability, susceptibility to inhibition
    • LDH1 in cardiac muscle H4
    • LDH2 in RBC H3M
    • LDH3 in lungs H2M2
    • LDH4 in other tissues HM3
    • LDH5 in liver and skeletal muscles M4
    • H4 higher affinity for substrate
    • heart attack→confirm dimeric CK tetrameric LDH (both 2 protomers) in serum by electrophoresis
    • heart muscle→CK1 BB, CK2 MB, CK3 MM
  • Multienzyme complex
    • catalytic domain→part of (more than one type) polypeptide chain the posses a catalytic function
    • >1 structural domain
    • a-ketoglutarate dehydrogenase→3 enzymes 5 coenzymes that catalyze oxidative decarboxylation of a-ketoglutarate, coenzyme A, NAD+ to succinyl-CoA, CO2, NADH in TCA
    • pyruvate dehydrogenase→3 enzymes 5 cofactors that catalyze oxidative decarboxylation of pyruvate with formation of acetyl-CoA, CO2, NADH
  • Holoenzyme=apoenzyme (responsible for reaction) + cofactor (bond formation between enzyme & substrate, transfer functional groups, form tertiary)
    • complete, functional enzyme
    • DNA polymerase 3, RNA polymerase
  • Metal ions
    • cofactors→Cu2+, Fe3+, Zn2+
    • Cd2+ and Hg2+ can replace Zn2+
  • Metalloenzyme
    • enzymes require metal ions
    • bind and retain metal atoms with very high affinity
    • metal-activated enzyme→low affinity for metal ion
  • Cofactor
    • catalytically essential molecules or ions covalently bound to enzyme
    • non-protein chemical compound/metallic ion as catalyst→vitamin, minerals, ATP
  • Coenzyme
    • organic molecules contain functionalities not found in protein
    • nonprotein with low molecular weight and heat-stable
    • NAD, NADP, FMN, FAD, lipoic acid, glutathione, coenzyme Q, iron porphyrins (heme), TPP, pyridoxal phosphate, CoA, biotin, tetrahydrofolic acid, ATP
  • Cosubstrate
    • coenzymes bind tightly to a protein, yet will be released and bind again at some point
    • NAD+, NADP+
    • alcohol dehydrogenase→ethanol + NAD+ to acetaldehyde + NADH + H
  • Prosthetic group
    • water soluble vitamin BC
    • malfunction of enzyme with lack sufficient cofactors derived from vitamin→dietary vitamin deficiency=functional & dietary
  • Specificity
    • stereospecificity
    • regioselectivity
    • chemo selectivity
  • 4 process
  • Lock key model→active site complementary in conformation to substrate
  • Induced fit model→binding site changes shape upon binding with specific substrate
  • Active site→pocket or cleft surrounded by aa side chains help bind substrate and by other side chains catalysis occurs
  • Serine protease
    • reactive serine residue at catalytic site→cleave peptide bond
    • chymotrypsin→hydrophobic residue=phe, trp, tyr
    • trypsin→positive charge residue/basic residue=lys, arg, his
    • elastase→specific for ala, gly, val
  • Enzyme kinetic
    • rate and mechanism of reaction
    • rate measure how many moles of reactant or product changed per time period
    • mechanism is how reaction occurs at molecular levels
    • rate of reaction depends on concentration of substrate [S]
    • rate = v = change in [P]/change in time = k[S]
    • rate is directly proportional to [S]
    • enzyme convert S to P→forward
    • as P grows, back reaction rate increases until equilibrium→reverse
    • graph not linear
      • as S larger, enzyme becomes limit
      • rate depends on fixed enzyme amount
      • rate asymptotically approach a max
      • rate depends on [S] but hyperbolic curve & plateaus (Vmax) depend on [enzyme]
  • Michaelis-Menten model
    • E+S to ES to activated ES* to EP to E+P
    • km=[S], high km=weak binding
    • Michaelis constant→Vmax /2
  • Lineweaver burk plot
  • Regulatory mechanism
    • [S]
    • [enzyme]
    • [cofactor/coenzyme]
    • [inhibitor/activator]
    • covalent modification
    • temperature→bell curve
    • pH
      • bell shape
      • alkalosis
      • acidosis
    • time
    • compartmentalization
  • Inhibitor
    • reversible
      • Competitive inhibitor block active site, Vmax same, high km
      • Non-competitive inhibitor + allosteric, alter active site, low Vmax, km same
      • Uncompetitive inhibitor + ES, low Vmax, low km
    • irreversible
      • enzyme inactivator
      • E+I=EI
      • take time to react with enzyme as slow formation of covalent bonds
      • time dependency→degree of inhibition increase with time
    • example
      • aspirin inhibit COX 1&2
      • allopurinol inhibit xanthine oxidase
      • ritonavir compete with substrate of HIV protease
      • tipranavir non peptide
      • methotrexate compete with dihydrofolate and substrate of dihydrofolate reductase, inhibit TMP synthesis, for leukemia
      • cyanide irreversible copper/iron in active site of cytochrome c oxidase
      • oligomycin inhibit ATP synthase
      • S-hexaglutathione
  • Nerve gas
    • diisopropylphosphofluoridate DIPF inhibit Ach ase
    • G agents
    • sarin
    • VX inhibit Ach ase, Na K pump open prolong, muscle contraction prolong

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