ENZYMOLOGY
• study of enzymes
Enzymes
• biological CHON’s that catalyze a certain rxn
catalyst – it makes the rxn faster
• essential to physiologic functioning:
 Hydration of CO2
 Nerve conduction - relying information from nerves
to brain
 Muscle contraction – for movement
 Nutrient degradation
 Energy use
Reactants:
• Enzymes – have two sites: active site and allosteric
site.
• Substrate – the one that binds to enzyme; should be
specific to its enzyme to have ES complex for them to
produce product.
* Enzymes are (1) bound within the cell and (2) they’re
specific in every organ such as cardiac enzymes, liver
enzymes, etc.
* If the cell or organ is degraded, the enzyme will
escape and will elevate its concentration to the
circulation /body.
Catalytic Mechanism of Enzymes
• A chemical reaction may occur spontaneously
if the free energy or available kinetic energy is
higher for the reactant than the
products(lower energy).
• Activation Energy – reactants have (enough) energy
to break their bond and collide to form new bond;
formed new bond is in between the enzyme and the
substrate to have chemical rxn.
Enzymes are highly specific:
Absolute Specificity – it is the strictest model; the
enzyme will combine only with 1 substrate and
catalyze only a single rxn.
Group Specificity – enzyme will combine with all
substrate with a particular chemical groups such as
ester group, carboxyl group, carbonyl group, etc.
Bond Specificity – similar with group spec. but with
all substrate with a particular chemical bond such as
ionic bond, covalent bond, hydrogen bond.
Stereoisometric Specificity – enzymes will combine
with substrate with particular optical isomer (same
number of C,H,O but with other position of one
molecule – “mirror image”).
DAEN
Factors that influence Enzymatic Reaction
1. Substrate concentration/Enzyme concentration
• follows the hypothesis of Michaelis & Menten –
focuses on enzymology.
“even in low substrate concentration, the substrate can
readily bind with free enzyme” – as long as the body
needs a fast chemical rxn, if the substrate is present
even in low concentration, it will still give a chemical
rxn.
Enzymatic Reaction can be:
 First-Order Kinetic – rxn rate is directly proportional to
the substrate concentration.
eg. 3 enzymes and 3 substrate – the body is too large
for the enzymes to easily bind with the substrate; add
more substrate for it to easily find its enzyme.
Zero-order kinetic – rxn rate is dependent only in
enzyme conc.
eg. add more enzyme to find its substrate (because
substrate conc. Is too low)
Saturation Kinetics – the maximum rate of rxn has been
reached (all are being locked and key), even if you add
more reactants it’ll not have any further rxn.
2. pH (power/potential of hydrogen)
• enzymes are protein that carry a net molecular
charge.
• enzymes will get denature/inactivate if placed in an
extreme pH.
 7 – 8 pH is the optimum for enzyme rxn but there are
specific enzymes who loves acidic pH such as acid
phosphatase and basic pH such as alkaline
phosphatase.
3. Temperature
• high temperature, high reactivity of an enzyme.
 40-50⁰C – causes denaturation of enzymes.
 60-65⁰C – inactivation of enzymes; catalytic function
of enzymes diminishes.
 37⁰C(25 or 30⁰C) – optimum temperature; most rxn
happens.
 Low temperature – can cause reversible inactivation
of enzymes, it’ll be active again once thawed;
however, repeated thawing will denature and
eventually will inactivate enzymes.
Temperature coefficient – every 10⁰C increased of
temp. will result in 2 folds increased reactivity of an
enzyme.
eg. 0 - 10⁰C = 2x increased; 10 - 20⁰C = 2x increased.

4. Co-factors
• non-CHON entities that must bind to an enzyme for a
rxn to occur.
 Activators – inorganic co-factors such as:
○ metallic - Ca, Fe, Mg, Mn, Zn, K
○ non-metallic - Br, Cl
• they alter the spatial configuration of an ezymes; it
alters the shape of an enzyme for the substrate to fit in
to have ES complex to have a chemical rxn.
Coenzyme – organic co-factors such as nucleotide
phosphatase, NAD and vitamins; it serves as second
substrate for enzymatic rxn; it also increases the
velocity of enzymatic rxn.
*binding site of enzyme to substrate is not match,
coenzyme will serve as “adaptor” for the enzyme so the
substrate can bind.
5. Inhibitors
• causes interference; it slows down or stops the rxn.
Types of Inhibition
Competitive Inhibition – inhibitor targets the active site
of an enzyme; it is match to the active site of an
enzyme and it competes with the substrate for it to
bind with the enzyme first.
Non-Competitive Inhibition – it targets the allosteric
site (site other than the active site) and will destroy the
enzyme for the substrate will not get bind; inhibitor is
not match to the active site.
Uncompetitive Inhibition - the target of the inhibitor is
the ES complex for it not to produce product.
6. Storage
• 20⁰C - for long-term preservation; freezing temp.
• 2 – 8⁰C – refrigerator temperature; general temp. for
preservation.
• Room temperature – 15 – 25 up to 30⁰C, used in cold
labile enzymes such as LDH4 & LDH5, they’ll get
denature in cold temperature.
7. Hemolysis – break down of RBC, enzymes will escape
through circulation like LDH; (LDH conc. is increased in
RBC).
8. Lactescence/Milky specimen – can cause decreased
enzyme measurement.
Enzymatic Reactions
• enzymes are present in very small amount in the
body; they’re measured in enzymatic activity not in
absolute value.
2 Methods in Measuring the Enzymatic Reactions:
 Fixed time/ End point – reactants are combined then
allowed to react in its designated time then rxn is
stopped and rxn is being measured.
DAEN
Continuous-Monitoring/ Kinetic Assay - multiple
measurement of rxn (placing the samples repeatedly
on water bath); being used to know if the product is
increasing or decreasing; used for enzyme
measurement.
Enzymes is a protein (Amino Acid)
• Primary Structure – specific amino acid (AA)
sequence.
• Secondary Structure – consists of polypeptide
twitching.
• Tertiary structure – formed by folding of polypeptide
chain (secondary structure).
• Quaternary structure – created in spatial relationship
between sub-units (tertiary structures being combined)
*The more complex the structure of the enzyme is, the
functional the enzyme function is.
Isoenzymes
• enzymes with same function/ catalytic rxn/ products
produced, but with different physical properties like
their charges, solubility, reasistancy.
eg. Creatine Kinase (CK) have isoenzymes CK1 (found in
brain), CK2 (heart) and CK3 (muscle).
They are differentiated by:
 Electrophoretic mobility – anode and cathode ends.
 Solubility
 Resistance to inactivation - using diff. chemicals.
Nomenclature
• Developed by the E.C. (Enzyme Commission)
1. Oxido-reductase – catalyze oxidation reduction
between 2 substrate.
2. Transferase – catalyze transfer of group other than
Hydrogen from 1 substrate to another.
3. Hydrolase – catalyze hydrolysis of various bonds; it
uses water to cleave compounds.
4. Lyase – catalyze removal of group from substrate
without hydrolysis (HoH).
5. Isomerase – catalyze interconversion of geometric
optical positional (GOP) isomers.
 6. Ligases – catalyze the joining of 2 substrate •Indicator of myocardial damge of acute myocardial
infarction (AMI)/ heart attack.
○ Rise= 4-8 hrs
○ Peak= 12-24 hrs
○ Normalize= 48-72 hrs.
*When having heart attack, it’ll rise then peak then
normalize.
*Other cardiac markers are LDH, AST, troponin and
myoglobin. Trop and Myg are CHON.
Other diagnostic significance:
• AMI
• Muscular Dystrophy
○ Duchenne Type – degenerative form of
muscular disorder; progressive loss of muscle;
have highest elevation of CK up to 50 – 100x than the
normal value of CK.
• CVA & other brain conditions
• Hypothyroidism, malignant hyperpyrexia, Reye’s
molecules. syndrome, Vibrio Vulnificus.
CREATINE KINASE (CK) *Value of CK may vary with gender, muscles mass, race
• Associated with ATP generation in contractile system (bc some may be naturally bulky, tall, etc.)
• Function: in the muscle cells it stores Creatine
Atypical Form of CK
phosphate that is important inATP production
Creatine phosphate + ADP ← CK →Creatine +ATP • abnormal mass
1. Macro CK
Tissue sources
• Found midway between MM AND MB in
1. Skeletal muscle – CK3 electrophoresis.
2. Heart muscle – CK2 • BB that is bound in IgG; LPP bound with CK-MM; not
3. Brain tissue – CK1 that clinically significance; seen in females who are
Isoenzymes: (dimer with 2 sub-units) more than 50 y/o.
CK-1 → <1% bc have short half-life; / CK- BB = brain 2. Mitochondrial CK (CK-Mi)
type. • Found before the MM
CK-2→ <6% / CK- MB = hybrid type (heart). • Bound to the exterior surface of mitochondrial
CK-3 → 94-100% / CK- MM = muscle type. membrane of the muscle, brain, and liver.
Indication: found in severe illness, malignant tumor,
Isoenzymes and cardiac abnormalities.
CK-MM
Methods used for the measurement of Isoenzymes of CK:
•Major fraction in serum.
•Elevated also in hypothyroidism (bc increased Electrophoresis – reference method.
permeability of the cell), muscle activity, IM (intra- Migration:
muscular) injection. Cathode Anode
CK-BB CK3 CK2 CK1
•Seldomly found in the plasma bc it only have 1-5 hrs Ion Exchange Chromatography – more sensitive but
half life, it can flushed out in our circulation right away. expensive.
•Have a high molecular size. • Problem with the bad column: CK-MM merge with
CK-MB CK-MBCK-BB eluted with CK-MB.
• most important isoenzymes. Antibodies- used specifically for ck mb determination/
•20% of cardiac tissue contains ck-mb, and very little to dx or AMI.
other tissues. • Anti-M antibody - inhibits all the activity of M
•Myocardium is the only tissue from which CK-MB subunit; removes all M sub-units.
enters the serum, meaning it is quite specific to the CK- BB CK- M CK- MM
heart muscle.
DAEN
 multiply the remaining B by 2 to measure the entire CK- 2. Immunoinhibition or chemical inhibition
MB, CK-BB have less interference in CK-MB 3. Substrate affinity – to see the affinity of alpha HBD
determination. (for measurement of LD1).
Immunoassay
• Detects MB reliably with minimal reactivity Diagnostic Significance:
• Detects enzyme protein rather than activity Elevated level: Renal, hepatic, cardiac, skeletal,
METHODS: hematologic, & neoplastic disorder.
• avoid hemolysis in CK determination bc there is AK
that can falsely elevate its concentration.
Storage:
• 4⁰C for 7 days
• -20⁰C for 1 month
*place it in dark place bc CK is photosensitive.

LACTATE DEHYDROGENASE
• Catalyzes the interconversion of lactic acid and
pyruvic acid.
Lactate + NAD ← LDH →Pyruvate + NADH
• Coenzyme: NAD
ISOENZYMES
Highest level of LDH: pernicious and hemolytic
Tetrametric molecules containing 4 subunits of two disorder.
possible forms. AMI: Rise: between 12-24 hours
i. LD1 - 14-26% = HHHH Peak: 48 - 72 hours
ii. LD2 - 29 – 39% = HHHM Normalize: after 10 days
iii. LD3 - 20-26% = HHMM
iv. LD4 - 8-16% = HMMM METHODS:
v. LD5 - 6-16% = MMMM 1. Wacker Method/ Forward or Direct rxn (8.3-8.9 ph
H = heart M = muscle @ 340 nm)
TISSUE SOURCES • lactate converts to pyruvate .
2. Wrobleuski and La Due/ Reverse or Indirect (7.1-
LD1 & LD2 = present in heart and RBC. 7.4pH @340nm)
LD3 = present in lungs, lymphocyte, spleen and • 3 times faster, small sample is needed
pancrease. • Susceptible to substrate exhaustion & loss of linearity
LD4 = present in liver.
LD5 = present in muscle. Reference Value: 100-225 U/L

*avoid hemolysis bc LDH conc. in RBC in 100 – 150 times

higher.
Storage:
• RT = read within 24 hours
• Cold temperatute = LD4 & LD5 will destroy; most
labile is LD5.

MEASUREMENTS OF ISOENZYMES
1. Electrophoresis – widely used and is combined with
flourometry or colometry to fully see the bands.
Part II
ASPARTATE AMINOTRANSFERASE (AST)
• Involved in the transfer of amino group between
aspartate and alpha keto acids.
Coenzyme: Pyridoxal Phosphate
REACTION:

DAEN
Aspartate + a-ketoglutarate ← AST →oxaloacetate + *amount of NAD is directly proportional to the conc. of
glutamate (glutamate – important in energy production ALT
of gluconeogenesis).
Isoenzymes: Storage: 4⁰C lasts for 3-4 days; not affected by
Cytoplasmic Isoenzyme hemolysis.
Mitochondrial Isoenzyme Reference Value: 6-37 U/L @37⁰C
*isoenzymes of AST are not routinely differentiated; it is
measured as total AST. ALKALINE PHOSPHATASE (ALP)
TISSUE SOURCES • Catalyze the hydrolysis of various phosphomonoester
at analkaline pH.
Cardiac tissue • It function to liberate inorganic phosphate froman
Liver - highest AST measurement is seen in acute organicphosphate ester with the concomitant
hepatocellular disorder. production of analcohol.
• viral hepatitis – AST conc. is increased up to REACTION:
100x. Phosphomoester + H20 (ALP at 9-10 pH) = alcohol +
• cirrhosis – AST is 4x increased. phosphate ion
Skeletal muscle - in muscular dystrophy and
TISSUE
inflammation, AST isSOURCES
4-8x increased.
*kidney, pancreas and RBC have small amount of AST. Liver
AST conc. in AMI: Bone
Rise: 6-8 hrs Placenta
Peak: 24-48 hrs Intestine
Normalize: 5th day post-infarction *small amount in kidney
METHOD: Karmen Method
•Aspartate + a-ketoglutarate ← AST →oxaloacetate + ISOENZYMES
glutamate Intestinal ALP
•Oxaloacatate + NADH + H Placental ALP
← MDH ( malate dehydrogenase )→malate + NAD Bone ALP
Liver ALP
Storage: Ref. temp – can last for 3-4 days; Avoid Placental isoenzyme - seen during 16-20 of gestation.
hemolysis. Intestinal isoenzyme - increased in type B or O after
Reference Range: 5-30 U/L @37⁰C consumption of fatty meal.
Bone isoenzyme - elevated in children during periof of
ALANINE AMINOTRANSFERASE (ALT) growth and adult ages >50 y/o because of the
• involved with the transfer of an amino group
osteoporosis.
fromalanine to a- ketoglutarate with the formation of
Liver isoenzyme - marker of hepatobiliary disorder esp.
glutamate and pyruvate.
biliary obstruction with an ALP measurement that is 3x
REACTION:
higher; this isoenzyme is present in sinusoids in bile
Alanine + a-ketoglutarate ← ALT →Pyruvate +
canaliculi.
glutamate
Liver isoenzymes bands:
TISSUE SOURCE o Major Liver Band
o Fast Liver Band ∝1 Liver Band – indicates
Liver - richest source that makes it a liver marker;
letastatic liver carcinoma; its fraction is very
with liver disorder, ALT is much higher than AST
small.
and it stays longer because ALT have 16-24 hrs
half-life.
Significance:
Obstructive jaundice; highest conc. of total ALP.
METHOD: Coupled Enzymatic Reaction (7.3 – 7.8pH
Paget’s disease/ osteitis deformans; highest
@340 nm)
elevation of bone ALP.
• Alanine + a-ketoglutarate← ALT →pyruvate +
*ALP is a liver and bone marker.
glutamate
*Activator of ALP is Mg.
• Pyruvate + NADH +H ← LDH →lactate + NAD
SEPARATION OF ALP ISOENZYMES
DAEN
 washing; >50 U/L is positive in rape case if seen
Electrophoresis in vaginal washing.
MIGRATION: Detection of Cancer – prostate tumor marker.
Cathode Anode
Intestine ALP Placental ALP Bone ALP Liver METHOD: Shinowara Method; at 5 pH
ALP Special consideration on enzyme activity:
Heat stability test – they are subjected at 56⁰C Prostatic ACP – inhibited by L-tartrate.
for 10-15 mins; most heat stable is I,P,L ALP and Red cell ACP – inhibited by cupric ion and
most heat labile is bone ALP. formaldehyde.
Chemical inhibition test Prostatic CA – coupled with PSA for its
o Phenylalanine – inhibits placental and intestinal determination.
ALP
o 3 molar urea – inhibits bone ALP Storage:
o Levamisole – inhibits liver and bone ALP At room temperature between 1-2 hrs, ACP will
*bone and liver ALP are co-migrator (they go in the decrease.
same band), add neurameridase to separate bands. 4⁰C – can last for 2 days
*avoid hemolysis; thrombocytopenia may increase ACP
Carcinoplacental ALP (abnbormal form od ALP, may
indicate malignancy): isoform of placental ALP. Reference value: 2.5 -11.7 U/L (total ACP)
Regan ALP – seen in lungs, breast, ovaries, 0-3.5 ng/Ml (prostatic ACP)
gynecological problem; heat stable up to 65⁰C
for up to 30 mins; a bone ALP co-migrator and is AMYLASE (AMS)
inhibited by phenylalanine. • It catalyze the breakdown of starch and glycogen.
Nagao ALP – seen in adenocarcinoma of • Smallest enzyme in terms of size; can filtered/pass in
pancreas, bile duct & pleural cancer; inhibited glomerulus hence, it is normal to have AMS in urine.
by N-leucine and phenylalanine. • Pancreatic tumor markers along with LPS, trypsin,
METHOD: Bowers & Mccomb Method (continuous chymotrypsin, and elastase 1.
monitoring technique); at 10.15 pH ISOENZYMES:
• p-nitrophenylphosphate← ALP →p-nitrophenol and  S-Type (s- salivary gland)
phosphate ion o Ptyaline
*rxn is inhibited by phosphorus o Anodal
 P-type (p- pancrease)
Reference value: 30-90U/L o Amylopsine
*ALP is 25% increased after high fatty meal o Cathodal
*avoid hemolysis because ALP is present in RBC Major tissue sources: salivary gland and pancreas
Other tissue sources: adipose tissue, fallopian tube,
Increased ALP small intestine, skeletal muscle
• Sprue – in intestine
• Osteomalacia Significance:
• Osteitis deformans in bones Acute pancreatitis
• Rickets AMS LPS
• Bone CA Rise: 2- 12 hrs 6 hrs
• Hepatitis and cirrhosis Peak: 24 hrs 24 hrs
• Hyperparathyroidism in liver Normalize: 3-5 days 8-14 days
• Obstructive jaundice Parotitis – increased AMS
Renal failure - increase of AMS to blood because
ACID PHOSPHATASE (ACP) glomerulus fails can’t excrete it.
Major source: Prostate Macroamylase – abnormal form of AMS; AMS that is
Minor sources: RBC, Platelets, Bone bound to Ig.
USES:
Forensic Chemistry – determines rape cases; Reference value: 60-180 SU/dL or 95-290U/L
activity of ACP lasts for 4 days in vaginal
DAEN
METHOD:
Inhibitor: wheat germ lectin,&TAG
Substrate: starch
1. Saccharogenic
• Measures the amount of reducing sugar produced by
the hydrolysis of starch by the usual glucose methods
by the action of AMS.
• Classic reference method expressed in SU.
2. Amyloclastic
• Measures AMS activity by following the decreases in
substrate concentration (degradation of the starch)
because of AMS.
3. Chromogenic
• Measures AMS activity by the increase in color
intensity of the soluble dye-substrate solution produced
in the reaction.
4. Coupled-enzyme
• Measures AMS activity by the continuous monitoring
technique.
• Commonly performed.
*AMS is increased in acute pancreatitis, mumps,
alcoholism, peptic ulcer and parotitis.
LIPASE (LPS)
• An enzyme that hydrolyzes the ester linkages of fats
to produce alcohol and fatty acid.
Major tissue source: Acinar cells of pancreas
Significance:
Acute pancreatitis – LPS in increased
Chronic pancreatitis – decline/reduction in LPS
METHOD: Uses olive oil and triolein; addition of
Colipase makes it more sensitive for acute pancreatitis.
Cherry Crandal method - hydrolysis of olive oil after
incubation for 24 hours at 37⁰C and titration of fatty
acids using NaOH; reference method determination.
TAG (in the form of olive oil) + H2O ← LPS→
monoglyceride and fatty acids
Reference value: 0-1.0 U/mL
*Miscellaneous enzymes*
ALDOLASE
• Splits fructose-1,6-diphosphate into two triose
phosphate molecules in the metabolism of glucose.
Isoenzymes:
Aldolase A – present in skeletal muscle
Aldolase B – present in WBC, liver, and kidney
Aldolase C - present in brain tissue
Increase level: skeletal muscle disease, leukemia, HA,
DAEN
hepatic carcinoimas.
5’ NUCLEOTIDASE (5’N)
‘ – prime
• marker for hepatobiliary disease; liver marker, liver
disease, and biliary disorder.
Reference value: 0-1.6 units
GAMMA GLUTAMYL TRANSFERASE (GGT)
• It catalyzes the transfer of glutamyl groups between
peptidesor amino acid through linkage at a gamma
carboxyl group.
Sources: liver (in canaliculi) , kidney, prostate &
pancreas
Sensitive indicator: alcoholism/occult alcoholism that is
seen in alcoholic person.
Elevated among individual: warfarin, phenobarbital, &
phenytoin therapy.
Highest value: Biliary tract obstruction
METHOD: Rosalki & Tarrow Method
Substrate: gamma- glutamyl-p-nitroanilide
Glutathione + amino acid >>(GGT) = glutamyl
peptide + cystalinyl glycine
Reference value: 5-30 U/L = Female
6-45U/L = Male
Cholinesterase/Pseudocholinesterase
• Used to monitor effect of muscle relaxant after
surgery; marker for insecticide and pesticide poisoning.
METHOD: Ellman techniques or Potentiometry
Reference value: 0.5-1.3 phunit
*sample is plasma
Angiotensin Converting Enzyme
• Indicator of neuronal dysfunction – alzheimer’s
disease; spx is CSF
• Sources: macrophage and epitheliod cells
Ceruloplasmin
• Copper carrying protein & an enzyme
• Hepatolenticular disease (wilsons’ disease)- decreased
value; liver marker
Glucose-6-Phosphate Dehydrogenase
(G6PD)
• Deficiency of this enzyme can lead to drug-induced
hemolytic anemia (antimalarial drug, primaquine).
• Increased in AMI, spx is serum.
Ref Value: 10-15U/g Hb or 1200-2000 mU/L per PRBC

Ornithine Carbamoyl Transferase

• Seen in hepatobiliary diaseas
Reference value: 8-20 mU/mL

DAEN