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Enzymes Cheat Sheet by

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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

Differ­ent­iation

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
 

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
 

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

Plot

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)
Ternary complex present

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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