ABZYMES, ISOENZYMES, RIBOZYMES

B. C. Muthubharathi2016501012

Abzymes Antibodies/Enzymes Catmab (catalytic Monoclonal antibody) Abzymes = Antibody +Enzymes

Similarity and Differences

Enzymes - bind in high energy state Antibody - binds the complementary structure

in its ground state ability to bind with compounds with great

specificity and high affinity

1986 - 1st monoclonal catalytic antibodies (abzymes) against a chemically stable analog of the transition state of a reaction were obtained

Peter Schultz and Richard Lerner Having structural complementarity for the transition

state of an enzyme catalyzed reaction They bind strongly to the transition state with high

association constant, enhancing the reaction rate Abzymes reduce rotational entropy

Sources

Do not occur naturally in human Found in normal human ( Anti Vasoactive intestinal

peptide autoantibody which detect the level of vasoactive intestinal peptide)

Natural abzymes in Patients with ADBlood of Asthuma – Reduce the rotational entropyAutoimmune diseased person (SLE)

Protoabzymes Proteolytic activity Found in AD patients – Asthuma, Multiple sclerosis, Thyroiditis,

Myocarditis

DNA abzymesDNA hydrolyzing activityPowerful regulator of apoptosis ( pathogenic role is

not clear)Cytotoxicity mechanism in systemic autoimmune

disease & Tumors

Production

Antibody molecules are produced by the immune system to bind and neutralize foreign substances called antigens

Foreign proteins of bacteria , viruses and some chemical molecules act as antigens

Transition state – State corresponding to highest potential energy along this reaction coordinates

Transition state analog- Molecules which are more stable than the transition state itself, but they mimic their 3D structure

Antibody Antigen Neutralize antigen

Foreign protein / HaptenTSA act as a hapten at TS and elicit antibody production, Antibody

isolated from serum used as abzymes

Substrate Transition stateProduct

Transition analog

Reactions of abzymes

Amide hydrolysis Trans

esterificationPhoto cleavage

OxidationDecarboxyl

ationCyclization

Hydrolysis of enol ethers

Reduction of diketone

Hydrolysis of hydroxyester Hydroxyester forms cyclic intermediate during hydrolysis

Hydroxyester Cyclic intermediate lactone Phenol

Increasing the reaction rate

Anti cyclic intermediate antibody

Cyclic phosphonate ester (Ag)( Mimic cyclic intermediate)

Hydroxyester forms cyclic intermediate during hydrolysis

Hydroxyester Cyclic intermediate lactone Phenol

Increasing the reaction rate

Anti cyclic intermediate antibody

Cyclic phosphonate ester (Ag)( Mimic cyclic intermediate)

Hydrolysis of ester Ester –Tetrahedral intermediate

P- nitrophenylacetate Tetrahedral p- nitrophenyl phosphate Acetic acid

intermediate

Binding (Catalysis)

Antibodies against phosphate analog of ester

phosphate analog of ester(Mimic the tetrahedral intermediate)

Pyridoxal 5’-P dependet aminotransferase reactionD alanine +pyridoxal 5’-p Pyruvate + pyridoxamine 5’ –p

Catalyze

Ab raised

N- (5’ phosphopyridoxyl)- lysineCoupled with carrier protein

Biosynthesis of heme ( Metallation) Introduction of ferrous ion into protoporphyrin

Ferrochelatase Mechanism – Distortion of pyrole ring by 36 to create bent transition

state

Protoporphyrin Bent transition state

heme (product)

N methyl mesoporphyrin Ab complementary (TSA) to TS

Activation of anticancer drug by abzymesThymidylate synthase Building block of DNA

5- fluorodeoxyuridasine-5 phosphateAbzymes

5- fluorouracil (Anticancer drug)

ISOENZYMES Same reaction but differ in aminoacid sequences, kinetic

properties and electrophoretic mobility

ONTOGENETIC – Same species – Intraspecific variants PHYLOGENETIC – Different species - Interspecific variants

HOMOLOGUES – Similar molecular structure &catalytic propertyEg.Phospholipase D

ANALOGOUS - Different molecular structure but catalyzing same reaction

Eg. Phenylalanine transaminase

List of isoenzymesLactate dehydrogenaseHexokinaseCreatin phosphokinasePhosphorylaseAlkaline phosphataseCytochrome P450G - 6 – P dehydrogenaseGlutamate dehydrogenase

Differ in amino acid composition and kinetic behavior so, by using electrophoretic method, isoenzymes can be separated

LACTATE DEHYDROGENASE Tetramer / oligomer (Different polypeptide chain) Four polypeptide subunit 2 types(H- Heart, M- Muscle) H4, H3M, H2M2, HM3, M4 (Catalyze the same reaction) Pyruvate + NADH + H+ Lactate + NAD

H4 LDH1 Heart MuscleH3M LDH2 RBC, Brain Muscle, LiverH2M2 LDH3 Brain,

LeucocyteHeart, Liver

HM3 LDH4 Leucocyte Heart, RBC,BrainMuscle, Liver

M4 LDH5 Muscle, Liver Other tissues

Increased level

Decrerased levlel

Glycolysis Pyruvate Lactate Blood

LDHLDH5 (M4) work best in NAD+ - regenerating directionBecause, activate muscle tissue become anaerobicHEART TISSUE

AerobicLactate as fuelLactate Pyruvate Citric acidCycleLDH1(H4) inhibited by excess pyruvate

DIAGNOSTIC ENZYME ( increased level)Heart attack, Hemolytic anemia, Muscle damage, Muscular

dystrophy, Stroke, Lung tissues, Death, hepatitis

Hexokinase 4 Isoenzymes I, II, III, IV I – Fastest mobility towards anode IV – Slowest mobility towards anode (also called as

Glucokinase) I – Brain, Heart, Kidney II – Skeletal muscle, Adipose tissue III – Liver, Spleen IV – Liver

D- Glucose + ATP D – Glucose + ADP

ANGIOTENSIN CONVERTING ENZYME 2 ISOENZYME

Somatic isoenzymesGerminal isoenzymes

Somatic – Lung, Vascular endothelial cells, Renal epithelial cells, Testicular epithelial cells, Testicular epithelial cellsGerminal – Sperm Regulate the blood pressure

Creatine phosphokinase 2 isoforms

B- BrainM- Muscle

Catalyze phosphorylation of crreatineCreatine + ATP creatine phosphate

CPK I – BB - Brain, LungsCPK II – MB – HeartCPK III – MM- Skeletal muscle

RIBOZYMESRIBONUCLEIC ACIDNot proteinAntisense RNAmolecules8 natural ribozymes1989 nobel prize, Thomas CzechCatalytic RNA moleculesRibonucleic acid enzymes/ RNA enzymes,RNAzymesRNA metaloenzymes~40 to 50 nucleotides in length

Sid Altman Tom Cech

separate catalytic substrate-binding domains Hammerhead, Hairpin for Therapeutic purpose Hammerhead ribozymes are preferable Ability to more efficiently recognize, Bind to, and cleave a range of different mRNAs. By altering the substrate-binding domain, a ribozyme can be

engineered to specifically cleave any mRNA sequence Inhibit the expression of a variety of viral genes and

significantly inhibit the proliferation of numerous organisms

Mang Yu &Coworkers Use ribozymes to provide WBC with resistance to HIV infection

In cell culture, ribozymes inhibit the expression of(1) human cytomegalovirus transcriptional

regulatory proteins, resulting in a 150-fold decrease in viral growth;

(2) human herpes simplex virus type1 transcriptional activator, resulting in a reduction of around 1,000-fold in viral growth; and

(3) A reovirus mRNA encoding a protein required for viral proliferation Hammerhead – treat collagen-induced arthritis in mice