ENZYMES

BY DR. MUDASSAR ALI ROOMI (MBBS, M. Phil)

ENZYMES
Introduction to enzymes ... Definition of enzymes (biological catalyst). Biochemical nature of enzymes (usually these are proteins but may be RNA in nature called as ribozymes).

PROPERTIES OF ENZYMES
Enzymes are biological catalysts that increase the velocity of reaction by lowering the energy of activation. Enzymes have active sites and may have allosteric sites. Catalytic efficiency. Specificity for their substrate (relative or absolute specificity). Enzymes usually need a non protein part for their normal activity. Holoenzymes = apoenzymes + non protein part. enzyme activity may be regulated in our body according to need. Enzymes are located within the cell in compartments.

NOMENCLATURE OF ENZYMES
Recommended names: these are simple names which are easy to remember and are commonly used in practice. These names usually end at the suffix ase attached to the substrate of the reaction e.g. lipase, sucrase, uricase and urease etc. some enzymes retain their original names which do not give any hint of the associated enzyme reaction e.g. trypsin and pepsin. Systematic names: this naming is difficult to remember and is according to the IUBMB nomenclature of enzymes (e.g. glyceraldehyde 3posphate NAD oxidodeructase).

Classification of enzymes according to IUBMB

Oxidoreductases: These enzymes catalyse the oxidation reduction reactions e.g. lactic dehydrogenase (LDH)and catalase enzymes. Transferases: These enzymes catalyze the exchange of groups between two compounds e.g. aminotransferases (AST and ALT). Hydrolases or hydrolytic enzymes: These enzymes catalyze hydrolysis i.e. decomposition of a compound and break it into two compoundsby addition of water into the substrate e.g. amylases, lipases, proteinases, sucrase, maltase, lactase. Lyases: These enzymes catalyze the removal of a group from a compound without the addition of water e.g. pyruvate decarboxylase. Ligases: These enzymes catalyze the reaction of joining two compounds by forming C=O, C-N, C-C, and C-S bonds e.g. pyruvate carboxylase. Isomerases: These enzymes catalyze the formation of isomers of the substrate e.g. phosphohexose isomerase.

DEFINE THE FOLLOWING TERMS

Holo-enzyme = Apoenzyme + non-protein part. Co-factor . Co-enzyme (Co-substrate or Prosthetic group). Lysozymes. Zymogens or pre-enzymes (e.g. pepsinogen, chymotrypsinogen).

ISOENZYMES OR ISOZYMES.
Definition: these are the enzymes having the same chemical properties but are different in their structure. Isoenzymes of LDH: LDH1, LDH2, LDH3, LDH4, LDH5. LDH1 is present in heart only and LDH5 is present in skeletal muscle only while the other isoenzymes are present both in heart and skeletal muscle. Isoenzymes of CPK: CPK-MB (in cardiac muscle), CPKBB (in brain tissue), CPK-MM (in skeletal muscle).

CO-FACTORS

CO-ENZYMES

HOW ENZYMES WORK

Template model or lock and key model. Induced fit model.

ENERGY CHENGES OCCURING DURING THE ENZYME CATALYZES REACTION

FACTORS AFFECTING REACTION VELOCITY

1. 2. 3. 4. 5. 6. 7. Effect of Substrate concentration. Effect of Temperature. Effect of temperature pH. Effect of enzyme concentration. Effect of product concentration. Effect of activators and coenzymes. Effect of modulators and inhibitors.

THE EFFECT OF SUBSTRATE CONCENTRATION ON REACTION VELOCITY MICHAELIS-MENTEN EQUATION????

This equation predicts how velocity of reaction(v) is related to substrate concentration (S) if enzyme concentration is held constant. v = Vmax(S)/Km+(S) Km is the substrate concentration at which velocity (v) is Vmax. Plot of velocity versus (S) is hyperbolic graph Just like oxygen myoglobin dissociation curve.

Q:

for a fixed amount of enzyme, what happens if you keep adding more and more substrate?

LINEWEAVER-BURK PLOT (DOUBLE RECIPROCAL PLOT).

Is called as double reciprocal plot because it is a plot between reciprocals of velocity of reaction(v) and substrate concentration (S). This plot is a straight line. Slope of this graph is Km/Vmax. The Y-intercept of the slope is 1/Vmax. The X-intercept of the slope is -1/Km.

INHITION OF ENZYME ACTIVITY

Competitive inhibition (competitive inhibitors are substrate analogs that compete with the substrate for the active sites of the enzyme ). Non-competitive inhibition (noncompetitive inhibitors bind at a site different from the active site)

QUESTION
What will be the effect of competitive and non-competitive inhibition on the graphs between v and (S); and graph between 1/v and 1/(S)??????

DIFFERENCE BETWEEN
Competetive inhibition
Reversible. Inhibitor and substrate resemble. Inhibitor bind the active site. Vmax is same. Km is increased. Inhibitor cannot bind with ES complex. Lowers substrate affinity to enzyme. Complex is E-I.

Non-competetive inhibition Not reversible. I and S don t resemble. binds other than active site. Vmax decreases. Km does not change. Inhibitor Can bind with E-S complex. Substrate affinity remains constant. Complex is E-I or E-I-S.

EXAMPLES OF COMPETETIVE INHIBITION IN BIOLOGICAL SYSTMEM

Statins (HMG Co-A Reductase inhibitor). Allopurinol (used to treat gout). Sulphonamides antibiotics (e.g. septran). Beta-lactam antibiotics (e.g. penicillins). ACE inhibitors (captopril,analpril and lisinopril). Anti-cholinesterase drugs ( e.g. Physostigmine and neostigmine). Warfarin.

Sulfa Drug Is Competitive Inhibitor

Para-aminobenzoic acid (PABA) Bacteria needs PABA for the biosynthesis of folic acid

H2NPrecursor

-COOH

Folic acid

H2N-

-SONH2
Sulfa drugs has similar structure with PABA, and inhibit bacteria growth.

Sulfa drug (anti-inflammatory drug)

EXAMPLES OF NON-COMPETETIVE INHIBITION IN BIOLOGICAL SYSTEM

Heavy metal inhibition of enzymes. e.g. lead can inhibit ALA- synthase and ferrochelatase enzyme in heme synthesis leading to anemia.

REGULATION OF ENZYME ACTIVITY

Allosteric regulation. Regulation of enzyme activity by covalent regulation. Induction and repression of enzyme synthesis by altering gene expression (by hormonal action).

ALLOSTERIC REGULATION
A low molecular weight substance (the effector or modifier) binds to the enzyme at a specific site other than the active site ( allosteric site) and increases or decreases its activity its activity (positive and negative effectors respectively). Allosteric enzymes usually have more than one subunit and more than one active site. In enzymes with multiple active sites that interact cooperatively, velocty (v) versus (S) plot is sigmoidal in shape just like oxygen hemoglobin dissociation curve.
Sigmoidal curve means binding of one substrate molecule facilitates or co-operates the binding of substrate at other sites (co-operative binding). When substrate itself acts as an effector the effect is called as homotropic. If the effector is different from the substrate the effect is called as heterotropic . Example 1: Heterotropic effetors (phosphofructokinase is allosterically inhibited by citrate). Example 2: Hexokinase enzyme is allosterically inhibited by glucose-6 phosphate.

Mechanism and Example of Allosteric Effect

Kinetics Models
Allosteric site

Cooperation
R

vo
S

(+)

Homotropic (+)

R
[S]

Allosteric site

(+) vo
S

A (+)

T
S

Heterotropic (+)

R
[S]

(-)

REGULATION BY CO-VALENT MODIFICATION

This is usually done by phosphorylation and dephosphorylation of the enzymes. Example 1: Glycogen phosphorylase (the enzyme that breaks down glycogen) is activated by phosphorylation. Example 2: Glycogen synthase (the enzyme that synthesizes glycogen from glucose) is activated by dephosphorylation.

INDUCTION AND REPRESSION OF ENZYME ACTIVITY

This is a long term process and is relatively slow to act. Usually required for growth or at other important physiologic events e.g. pregnancy. This process is usually hormone dependent which act on DNA to affect its transcription into RNA.

ENZYMES IN CLINICAL DIAGNOSIS

Alteration of plasma enzyme level in disease state. Plasma enzymes as diagnostic tools. Isoenzymes and diseases of the heart(CK, TROP-T and TROP-I). Amylase: raised in parotitis and acute pancreatitis. Acid phosphatase: diagnostic for prostatitis and prostate cancer. Alkaline phosphatase (ALP): diagnostic for obstructive jaundice. Gamma glutamyl tranferase: diagnostic for obstructive jaundice. AST and ALT : these are diagnostic for acute hepatitis. LDH: Is diagnostic for acute myocardial infarction. CPK: Is also diagnostic for acute MI.

ENZYMES USED TO DIAGNOSE HEART ATTACK

CPK-MB: peaks within 24 hours and usually comes back to normal before 48 hours. TROP-T and TROP-I(Newest markers for diagnosis of MI): These enzymes peak within 8-28 hours after heart attack and remain elevated for 3-10 days. AST or SGOT: peaks at 24-48 hours and may fall back to normal by 72 hours. LDH-1: Peaks at 3-4 days and remains elevated for 10-14 days.

ENZYMES AS LABORATORY REAGENTS

Glucose oxidase: to detect glucose in urine and blood). Uricase: to detect uric acid in blood and urine Urease: to detect urea in blood and urine.

THERAPEUTIC USES OF ENZYMES

Streptokinase and urokinase (for lysis of blood clot e.g. after heart attack). L-asparaginase (for the treatment of lymphoblastic leukemia). Digestive enzymes as supplements in cases of digestive disorders e.g. amylase, lipase,and protease. Trypsin and Chymotrypsin (for degradation of necrotic tissue). Hyaluronidase (for better absorption of ointments).