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44 Cards in this Set

  • Front
  • Back
3 common features of proteases
activate H2O or other nucleophile
polarize peptide carbonyl group
stabilize tetrahedral intermediate
chymotrypsin
digestive enzyme; protease that cleaves peptides at carboxy terminus of hydrophobic amino acids like tryptophan, tyrosine, Phe, and Met (in presence of water)
enzyme is acylated and X is released forming Acyl-enzyme intermediate then enzyme is deacylated with water and enzyme is restored
structure of active site in chymotrypsin
side chain of Ser is H-bonded to His and NH group of His is H-bonded to carboxylate group of Asp--referred to as catalytic triad
carbonic anhydrase active site
Zn2+ atom bound to 3 His imidazole rings and one water molecule
mechanism for carbonic anhydrase
Zn2+ bound to H20 decreases pKa to 7, so loses H+. OH- attacks CO2
what do nucleoside monophosphate kinases do?
catalyze transfer of terminal phosphoryl group of a nucleoside triphosphate (usually ATP) to the phosphoryl group of a nucleoside monophosphate
what is the substrate for nucleoside monophosphate kinase reactions?
divalent metal--Mg2+ or Mn2+ (almost all NTPs are bound to one of these); usually bound to 2 O's of NTP + some water molecules
what does the magnesium ion do to the substrate/enzyme in NMP kinase reactions?
provides additional points of interaction btw ATP-Mg2+ and enzyme; coordinates the beta and gamma phosphates so that the gamma phosphate can be transferred to NMP
what is the p-loop on nucleoside monophosphate kinases? what does it do?
loop between beta strand and first helix; interacts with phosphoryl groups on bound nucleotide
name of the mechanism used by NMP kinases
catalysis by approximation
what metal ion is necessary for restriction enzymes? What does it do?
Mg2+; bound to 3 water molecules, 2 carboxylates of enzyme's Asp residues, 1 to O of the phosphoryl group at site of cleavage; holds water molecules in place so that it can attack phosphoryl group, and (with Asp) polarizes H2O molecule toward deprotonation
classic example of induced fit
nucleoside triphosphate kinase reactions; binding of substrate induces large structural changes in kinase
what are the changes induced by the binding of substrate in NMP kinase reactions?
P-loop closes down on top of polyphosphate chain, movement of P-loop brings down top domain of enzyme to form lid over bound nucleotide; ATP is held in position with gamma phosphoryl group positioned next to binding site of NMP
covalent catalysis
active site contains reactive group (usually powerful nucleophile) that becomes temporarily covalently bonded to substrate during reaction (i.e. chymotrypsin)
proteases (what do they do and 4 types)
cleave proteins by hydrolysis--add molecule of water to a peptide bond; aspartyl, serine, cysteine, metalloproteases
covalent catalysis
active site contains reactive group (usually powerful nucleophile) that becomes temporarily covalently bonded to part of substrate during reaction
chymotrypsin has 2 phases to reaction--what are they, why?
"burst" and steady-state; burst is first step of reaction--faster than second step, which is stead-state release of substrate
roles of residues in catalytic triad in chymotrypsin
His-positions Ser side chain and polarizes hydroxyl group so poised for deprotonation (in presence of substrate, it takes proton)
Asp-orients His residue, makes it a better proton acceptor
Ser-binds to substrate
Gly-also important--stabilizes oxyanion hole
2 other catalytic triads
trypsin (replace Ser with Asp--attracts positively charged side chains)
Elastase (2 Val residues at top of active site limit size of side chains)
example of a protease inhibitor used as a drug
HIV treatments inhibit HIV protease (an aspartyl protease)
catalysis by approximation
brings substrates into proximity at proper orientation
nucleoside monophosphate kinases
catalyze transfer of terminal phosphoryl group from a nucleoside triphosphate to phosphoryl group of nucleoside monophosphate
What structural motif do NMPases have?
p-loops (between first beta strand and first helix)
restriction enzymes
catalyze hydrolysis of phosphodiester backbone of DNA (bond between 3' O atom and phosphorous atom is broken)
structure of restriction enzymes
twofold rotational symmetry--enzyme surrounds DNA tightly
host DNA can't be distorted by restriction enzymes because...
methyl groups on adenine bases prevent H-bonding to Asn residue in enzyme
distortion of DNA increases binding energy by....this is canceled out by....
increasing points of contact; distortion
aspartate transcarbamoylase
catalyzes first step in biosynthesis of pyrimidine nucleotides like cytidine triphosphate; has T and R state (expands on substrate binding)
structure of ATCase
2 catalytic trimers stacked on top of eachother, 3 regulatory dimers link catalytic subunits together
PALA
resembles catalytic intermediate (bisubstrate analog) of ATCase, so inhibits it--stabilizes R state
mechanis for allosteric regulation of ATCase is
concerted--changes in enzyme are all or nothing--either in T or R state (not like hemoglobin=sequential)
CTP
product of ATCase reaction--inhibits reaction by stabilizing T state--higher apparent Km; ATP lowers apparent Km
isozymes
enzymes that differ in sequence but catalyze same reaction; lactate dehydrogenase
lactate dehydrogenase
catalyzes a step in anaerobic glucose metabolism/synthesis; humans have 2 isozymic polypeptide chains (H and M); can have different combinations--H3M
protein kinases
transfer phosphoryl groups from ATP (usually) to Ser, Thr, Tyr; turn on signaling pathways
protein phosphatases
reverse effects of protein kinases; turn off signaling pathways
effects of phosphorylation (6)
1) adds 2 neg. charges to protein
2) allows formation of new H-bonds
3) energetically favorable
4) reaction rates can be adjusted by [E] and Vmax
5) amplifies effects--activates many other enzymes
6) ATP is cellular energy currency--links energy status to metabolism
trypsin
activates all pancreatic zymogens; cells that line duodenum secrete an enzyme that hydrolyzes peptide bond in trypsinogen to activate it
pancreatic trypsin inhibitor
6kDa protein; binds very tightly to active site of trypsin
intrinsic vs. extrinsic clotting pathways
intrinsic: occurs due to rupturing of endothelial lining of blood vessels
extrinsic: occurs due to trauma-related damage of blood vessels (exposure of tissue factor)
thrombin
activates enzymes and factors that lead to generation of more thrombin, converts fibrinogen to fibrin
fibrinogen
made up of 2 alpha, beta, gamma chains; thrombin cleaves alpha and beta chains to make fibrin; cleaved ends of alpha/beta chains associate with globular domains of beta and gamma to clot
gamma carboxyglutamate
binds Ca2+ in Gla domain of prothrombin
inhibitors of Vit. K-dependent pathway of thrombin
warfarin, dicoumarol