Lactate dehydrogenase
lactate + NAD+ --> pyruvate + NADH + H+
tetrameric
H-form: aerobic , heart L-->P
M-form: anaerobic , muscle/liver P-->L
Reagents: Lactate,NAD+,Oxidized PMS, Oxidized NBT
Specific Activity stain
LDH-1: pyruvate inhibition
LDH-1/2: 2-hydroxybutyrate as S
LDH-4/5: greater heat stability |
Creatine Kinase
Creatine + ATP <--> creatine phosphate + ADP + H+
Dimeric
Cardiac: MM+ MB (Myocardial infarction)
Skeletal: MM
Brain: BB |
Chymosin (Rennin)
Aspartic protease
Cleave single peptide bond,
release acidic C-terminal peptide
Ca induced aggregation of modified casein micelle--> precipitate as curd |
Affinity label
Specific & Irreversible inhibitor
Specificity group & reactive group
resembles substrate
TPCK on His-57 of Chymotrypsin |
Determination of enzyme activity
NAD+ : absorbance change at 340nm
FAD: absorbance change at 440 nm X |
Deter
Follow INITIAL rate, rate drops
1. substrate depletion
2. reverse reaction
3. product inhibition
4. enzyme stability |
Homeostatsis(regulation)
1. [S] control, M-M vs Cooperativity
2. Allosteric effect
3. [S]cycle, 2-way, 6-Phosphofructokinase & Fructose bisphosphatase
4. Zymogen activation
5. Covalent modification (phosphorylation,adenylylation,myristoylation,ADP-ribosylation,methylation,acetylation,ubiquitination)
6. Enzyme cascade
7. Cascade amp
8. Enzyme induction/degradation |
Modification of amino acids
Active site residues more susceptible
Ser-195 on chymotrypsin by DIPF -->Activity
Modify 1st with [S]/[I] to protect active site, then modify again in absence
ADH inactivated by iodoacetate more than iodoacetamide
2AA involved: pKa >2 units apart:Good
Close--> tVmax never achieved |
Lite Beer
Barley:α-amylase cannot break down α-1,6 bond + dextrin --> Yeast
Glucoamylase from Aspergillus niger: break α-1,6 bond, less dextrin |
Aspartame
Thermolysin
L-phenylalanine + N-protected L-aspartate
N: benzyloxycarbonyl |
Catalysis
1. Strain/Distortion: entropy reduction
2. Acid-Base : carbonic anhydrase
3. Covalent catalysis: serine protease
4. Lower Ea: Zn&Arg127 in carboxypeptidase A stabilize TS |
Histidine can be both e- donor/acceptor
TS analogue: pyrrole-2-carboxylate on proline racemase as inhibitor
Abzyme: mimic Ferrochelatase
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Ribozyme
RNA less versatile (4 building blocks AUCG)
unable to form large non-polar region
nucleic acid preferred as substrate
RNA susceptible to hydrolysis
Organic Cofactors
Catalytic cofactor: e.g. TPP/FAD
Stoichiometric cofactor: cosubstrate
Radioisotope
Glutamate decarboxylase
CO2 : trap gas in alkali
Monoamine oxidase
R-CHO:extracted by ether after acidification (acidified R-NH2 will remain in aq phase)
Cholinesterase
COOH: ion exchange, importance of label position |
Scintillation Proximity Assay
Radioligand stimulate bead to emit light, when in close proximity
High affinity capture system: biotinylated substrate & streptavidin-coated beads
NO separation needed. S or P bind to bead |
Competitive inhibitor
Same site, mutually exclusive
Vmax unchanged Km increased
Non-competitive inhibitor
ESI present, Km unchanged, Vmax decrease,
equal Ki,same % inhib.
Pre-steady state kinetics
E+S--> ES(flurorescence)
Stopped flow technique, follow time course of fluorescence change |
Irreversible inhibitor
Diisopropyl phosphofluoridate (DIPF) modifies serine on AchE |
Hill Coefficient
Important: Choice of [S]
Cooperativity: Same site&ligand
Chymotrypsin
Active site Ser195,His57&Asp102 form charge relay system--> high reactivity of Ser195
Selective for carboxyl side of aromatic or large hydrophobic residue(Met)
Biphasic kinetics:
1. Burst phase: covalent complex
2. SS Phase: hydrolysis+ recovery
Double displacement, p-nitrophenolate |
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Cofactor
Apoenzyme |
inactive form |
Cofactor |
non-protein |
Holoenzyme |
active |
Metalloenzyme |
participate in reaction(lewis acid) |
Zn in carbonic anhydrase |
Metalloenzyme |
stabilize transition state |
Zn in carboxypeptidase A |
Metal activated enzyme |
maintain active conformation |
K+ in pyruvate kinase |
Metal activated enzyme |
form substrate complex bridge |
Mg in kinase |
Prosthetic group |
tightly bound |
NH2 on pyridoxal phosphate of aspartate transaminase |
Coenzyme |
loosly bound |
Cosubstrate(coenzyme) |
convert to product after Rx |
NAD+ |
Coenzyme analogue as drug
Drug |
Analogue of |
enzyme inhibited |
MOA & Use |
Sulfonamide |
PABA |
dihydropteroate synthase |
folic acid synthesis,Abx |
Methotrexate |
Folate |
dihydrofolate reductase |
THF synthesis,childhood leukemia |
Uncompetitive inhib.
S binding to E--> expose site for I binding
both Km Vmax decrease to same extent, ESI present, same slope |
Mixed inhibition
Binding affinity(Ki) not the same
Vmax decrease, Km can in/decrease |
Suicide substrate
P irreversibly bind to E
Deprenyl on MAO on Flavin prosthetic group |
Substrate inhibition
High [S] favour ESS(nonproductive binding)
e.g. succinate dehydrogenase (select points for drawing) |
Single displacement Rx
Random sequential |
creatine kinase |
Compulsory order |
ADH(NAD+ bind 1st) |
Double displacement Rx (Ping-Pong)
aspartate transaminase
aspartate+ α-ketoglutarate -->oxaloacetate+ glutamate (NH2 displaced) |
Isotope exchange
Occurs only in double displacement;
exception: maltose phosphorylase
isotope from 1st P back to 1st S in absence of 2nd S e.g. sucrose phosphorylase
Glu-Fru + Fru* <--> Glu-Fru* + Fru |
Diff. subunits of multimeric enzyme
Catalytic & Regulatory |
Aspartate transcarbamoylase(ATCase) |
ATP&CTP |
2nd unit modify specificity |
Lactose synthase |
α-lactalbumin |
2 diff. cat. units |
tryptophan synthase(α2β2) |
Tunnel connect active sites |
Lysozyme
hydrolyze glycosidic bond bet C-1 of NAM and C-4 of NAG, Non-identical site
Site of cleavagea: bet D&E, distorted Ring D
Glu-35 as acid, H+ to O of glycosidic bond
Carbonium cation stabilized by
1. -ve charge on Asp-52
2. half-chair formation of sugar D (strain) (resonance stabilize +charge on C-1 with O) |
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