Lecture Enzymes

7/28/2019 Lecture Enzymes

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ENZYMES

Siv Milliscent E. Balbas, RMT


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What are Enzymes?

CHON catalyst of chemical reactions andcatalyzes all metabolic reactions of cells

(eg. Respiration, photosynthesis, digestion,)

biological catalyst

specific for its substrate


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Parts of an Enzyme

Active site- portion of the enzyme wherereaction takes place

Substrate- binds to Active site of an enzyme

Allosteric site Other site besides the active site


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ENZYME

SUBSRTATE

LOCK AND KEY FIT


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ENZYME

SUBSRTATE

INDUCED FIT


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Cofactor

Nonprotein molecule which maybe necessary forenzyme activity

Coenzyme- Organic cofactor (NAD, heme,

vitamins)

Activators- Inorganic cofactor (eg. Chloride or

magnesium ions, and other metals)


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ENZYME

SUBSRTATE

Cofactor

HOLOENZYME

APOENZYME


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NOTE:

Holoenzyme- complete, active enzyme system Proenzyme (zymogen)- inactive enzyme

Isoenzyme- enzymes that have differentphysical properties but have the same

catalytic functions


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Classification of Enzymes

OXIDOREDUCTASES

Catalyze an oxidation-reduction reaction between2 substrates

TRANSFERASES

Catalyze the transferof a group from one

substrate to another


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HYDROLASES

Catalyzes hydrolysis of various bonds

LYASES

Removalof groups without hydrolysis

ISOMERASES Catalyze the interconversion of geometric, optical

or positional isomers

LIGASES Catalyze the joining of two substrate molecules,


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Factors the Influence

Enzymatic Reactions SUBSTRATE CONCENTRATION

pH ( 7.0- 8.0)

TEMPERATURE

ENZYME CONCENTRATION

COFACTORS

INHIBOTORS Competitive inhibitors

Noncompetitive inhibitor

Uncompetitive inhibitor


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Competitive Inhibitors

Competes with substrate for binding to active site

Non-Competitive Inhibitors

Does not bind to the active site, but removes

cofactors of the enzyme

Uncompetitive Inhibitors

Inhibitor binds to E-S complexpreventingdissociation


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Enzyme Tissue Specificity

High Moderate Low

ACP(RBC, Prostate)

AST(Liver, heart, skeletal

muscle))

LD(In ALL tissues)

ALT(Liver)

CK(Heart, skeletal muscle,

brain)

AMS(Pancreas, salivary glands) ALP(liver, bone, kidney)

LPS(Pancreas)


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Hepatic Enzyme Profile

ALP

AST

LD (LDH)

GGT

ChE


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Alkaline Phosphatase (ALP)

optimum pH : 10

highest concentration in liver, kidney,placenta, intestine, WBC


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Increase levels in:

Bone formation

Pagets disease

Rickets

Osteoblastic tumors

Cancer

Sever fracture

Liver disease


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Collection:

AVOID using citrate, oxalate, EDTA

Substrate: Organic phosphates (eg. -glyceroPO4 and -nitrophenylPO4)

Methods:

Bodansky(-glyceroPO4)

Shenowara Jones (-glyceroPO4) King- Armstrong (-nitrophenylPO4)

Bessy Lowry-Brock(-nitrophenylPO4)


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Alkaline AminoTransferase

(ALT) Serum Glutamic PyruvicTransaminase (SGPT) Found in the LIVER and RBCs

More specific for liver than AST

Marked elevation with viral hepatitis

Collection: AVOID HEMOLYSIS


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Catalyzes the reaction:

Alanine + ketoglutaric acid Pyruvic Acid

Method:

Reitman Frankel

Addition of DNPH (2,4- dihydrophenylhydrazine) topyruvic acid to produce color

ALT


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ALT (Liver)

AST (Heart)Glutamate

oxalacetatetransaminase

Substrate Alanine - Keto Aspartate keto

End Product Pyruvic Acid Oxaloacetic Acid

Location Heart, LIVER HEART, Liver

HighConcentration

LIVER HEART

Significance Liver DiseaseMycocardial

Infarction


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ALT/AST RATIOde ritis ratio

Differentiates VIRAL from NON-VIRAL etiology

Viral Etiology: High ALT If ALT/AST is >1 = VIRAL

Non-viral: High AST If ALT/AST is


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Lactate DeHydrogenase

(LD/LDH)

Catalyzes reversible lactate pyruvate

using NAD+

as a cofactor

5 isoenzymes (electrophoresis):

LD1, LD2, LD3, LD4, LD5


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4 SUBUNITS in each Isoenzyme

ISOENZYME SUBUNITS SHORTHANDSUBUNITS

HIGHCONCENTRATION

LD1 HHHH LD H4 Heart, RBC, Brain

LD2 HHHM LD H3M Heart, RBC, Brain

LD3 HHMM LD H2M2Brain, Kidney,

Lung

LD4 HMMM LD HM3 Liver, Skeletalmuscle, kidney

LD5 MMMM LD M4Liver, Skeletalmuscle, ileum


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Collection: AVOID HEMOLYSIS

clot must be separated from serum

quickly to prevent increase in LD1 and LD2

Storage: 25C upto 24 hours

Only LD4 and LD5 are utilized to test LIVERDISORDERS. May also be elevated duringskeletal muscle damage


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Gamma-Glutamyl

Transferase/Transpeptidase

(GGT)

Increase in liver disease, metastaticcarcinoma , alcoholism and hepatobiliary

obstruction

Marker for daily consumption of largeamount of alcohol or drugs (barbituates,

phenytoin)

Method: SZAZ- substrate: gammaglutamyl

-nitroanilide


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Pseudocholinesterase (ChE)

Cholinesterase (true and Pseudo)

Cleaves Acetylcholine: the bodys majorneurotransmitter

True ChE

high activity in the CNS, RBC, lungs and spleen.

Pseudo-ChE or acylcholine acylhydrolase primarily produced in the liver

Also produced by myocardium and pancreas

Important in cleaving SUCCINYLCHOLINE


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Significant decrease seen after exposure tophosphorus compounds found in insecticide,

nerve gases and pesticide.

Also seen in anesthetic poisoning.

Substrate: acetylcholine

Method:

Michael

Ellman


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CARDIAC DISORDER PROFILE

CK-MB

AST

LD (LDH) 1 & 2

LD/HBD ratio

MI Biomarkers


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Creatine Kinase (CK)

found in cardiac, skeletaland brainmuscle

Has 3 isoenzymes:

CK-BB (CK1)= Brain

CK-MB (CK2)= Heart, muscle

CK-MM (CK3)= Muscle, heart


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Catalyzes

phosphocreatine + ADP creatine + ATP

Methods:

Tanzer-Gilvarg: (forward)

Phospho Kinase + LD Lactate + NAD

Oliver- Rosalki: (reverse)

Hexokinase + G6PD 6-P Gluconate + NADPH


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ASpartate aminoTransferase

(AST)

Most sensitive enzyme foe skeletal muscle

disease

Collection: AVOID HELOLYSIS

Inhibited by all anticoagulants except heparin(do not use ammonium heparin)


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Lactate DeHydrogenase 1 & 2

LD 1 (Anodic), while LD 5 is (cathodic)

LD 1 and LD2 (HEAT STABLE)

LD5 (COLD LABILE)

Used in evaluating Cardiac Disorders


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Normally LD1< LD2

Normal Ratio is 0.5-0.75 or LD2 it is called a flipped ratio Flipped ratio is seen in MI (provided sample is not

hemolyzed)

50% MI: flipped ratio in 48hours 80% MI: flipped ratio in 72 hours


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LD/HBD(hydroxybutiric

dehydrogenase) Ratio

Normal LD/HBD ratio: 1.2- 1.6

If ratio is 0.8-1.2, MI is suspected


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Non-Enzymatic MI Markers

MYOGLOBIN Major protein responsible for oxygen supply of

striated muscle

TROPONIN the troponin complex is a component of the thin

filament of striated muscle linked to actin

Three Subunits: Troponin I: an inhibitory subunit

Troponin T: tropomyosin-binding subunit

Troponin C: Calcium-binding subunit


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ACUTE PANCREATITIS PROFILE

AMYLASE

LIPASE


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Amylase

found in the SALIVARY GLANDS andPANCREAS

Breaks down starch to simple sugars

Substrate: starch

starch/ amylum maltose glucoseAMS Maltase


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Substrates:

Pancreatic AMS: diastase

Salivary AMS: ptyalin

Serum AMS is usually pancreatic in origin

microAMS: unbound, free. 50,000 dal. AMS

found in urine

macroAMS: bound to IgG, IgA. High MW.

Measured in serum


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Methods

SaccharogenicMeasures amount of maltoseproduced (glucose: Somogyi)

Iodometric/amyloclastic Measures starch remaining

ChromogenicMeasures dye released frombreakdown of polysaccharide

Kinetic Method Measures change of NAD toNADH at 340nm


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Lipase

Breaksdown Triglyceride into fatty acids andglycerol

Significance: Acute Pancreatitis

Substrate: Olive Oil

End Product: Fatty Acids

Methods: Cherry-Crandall

Sigma-Tietz

Titration


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PROSTATIC CANCER PROFILE

ACP

PSA


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ACP (pACp)

Optimum pH: 5

Very Labile, Add 5M acetate buffer or citrate

tablet to preserve

ACP are found in Prostate, RBC, bone, liver,kidneys, platelets

Substrate: organic Phosphate such as-glyceroPO4 and -nitrophenylPO4


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Method

Chemical Inhibition Test

If Total ACP is normal: Stop test

If elevated: suggestive of prostatic CA

do p-ACP by Chemical Inhibition Test

Cu++ Tartrate C4H4O6-2

RBC-ACP Inactivated (+) Unaffected (-)

p-ACP Unaffected (-) Inactivated (+)


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PSA (Prostate-Specific Antigen)

Member of the kallikrein familyof sereinproteases uniquely produced form theepithelial cells of the prostate gland

Most useful TM for Prostate Cancer

Drawback: weak in distinguishing prostate CA

from nonmalignant prostate lesions

Reference range: 0-4ng/mL


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Good luck and God bless!

Study well and Pray Harder!

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Lecture Enzymes

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