- Post-translational modification (PTM) refers to the

ENZYMES covalent and generally enzymatic modification of

proteins during or after protein biosynthesis.
- proteins that function as biological catalysts COFACTOR
- Catalysts - non CHON molecule of enz necessary for enz activity
- substance that usually speed up the rate of a chemical - activators: inorganic cofactors (Mg2+)
reaction and is not changed by the reaction - coenzymes: organic cofactors (NAD)
- biologic proteins - prosthetic group - tightly bound coenzyme
- catalyze chemical and many physiologic reactions - apoenzyme - enzyme portion
- important for physiologic functions
- found in all body tissue apoenzyme + prosthetic group = holoenzyme
- increase in enzyme = cellular damage
- thus enzymes speed up the rate of metabolic reactions in Holoenzyme – are complete and active system.
the cells
- hydration of carbon dioxide, nerve conduction, muscle proenzyme or zymogen
contraction, nutrient degradation for energy use - an inactive form of enzyme (pepsinogen - pepsin: active)
- enzymes usually appear in high concentration following
cellular injury ENZYME CLASSIFICATION
OXIDO REDUCTASE
ENZYME STRUCTURE - catalyzes REDOX
Primary TRANSFERASES
- amino acid seq - transfer of group other than hydrogen from one substrate
Secondary to another
- polypeptide twisting HYDROLASES
Tertiary - hydrolysis of various bonds
- polypeptide folding LYASES
Quaternary - removal of groups from substrate w/o hydrolysis; the
- spatial relationship between 2 or more polypeptides product contains double bonds
ISOMERASE
active site: water free cavity, where substrate attaches, where - interconversion of geometric, optical or positional isomers
catalytic activity occurs LIGASES
- joining of 2 substrate molecules
allosteric site: bind regulator molecule ; non covalent,
reversible TYPES OF SPECIFICITY
ABSOLUTE SPECIFICITY
reactant: substrate, specific for particular enzyme - one substrate only
enzyme substrate complex: binding brings key atoms near GROUP SPECIFICITY
each other and stresses key covalent bonds - one chemical group (phosphate esters)
BOND SPECIFICITY
ENZYME KINETICS - specific type of bond
Reactants to products - reactants should have higher energy STEREOISOMERIC SPECIFICITY
than barrier - one type of isomer
Activation energy - excess energy needed by reactant to
break chemical bonds to form new bonds FACTORS INFLUENCING ENZYME ACTIVITY
Ways to allow product formation Substrate concentration
- provide more energy by increasing the temp - first order kinetics (Michealis and Menten)
- use enzyme to lower energy barrier - reaction rate id directly proportional to substrate
concentration
Enzymes are catalysts: - first order: enzyme is in excess
This means that enzymes help speed up chemical reactions.
How? Enzyme concentration
- Enzymes lower the activation energy of a reaction. - zero order kinetics
- If the Ea is lower, more reactant molecules will make it over - increased ENZ level, faster reaction
the “energy hill”, so more products are produced per - excess substrate therefore reaction will depend on
second. enzyme activity, the more enzyme activity, the higher the
speed of reaction
TERMS
SUBSTRATE- Substance in which enzyme acts pH
ACTIVE SITE- where substrate interacts with particular - 7.0-8.0
charged amino acid residues - changes may result to denaturation
ALLOSTERIC SITE- binds the regulator molecules - except for specific enzymes that are active in low pH or
ISOENZYME high pH
- enzymes with same function but exist in different forms in
the body temperature
- can be differentiated from each other, based on their - increase in temp causes increase in reaction
physical properties like electrophoretic mobility, solubility,
resistance to inactivation. cofactor
ISOFORM- results when enz are subjected to post - provided in excess so reaction doesn’t depend on
translational modifications concentration of cofactor
inhibitors
- competitive, on competitive, uncompetitive ATYPICAL CK
Mitochondrial CK
competitive - bound to the bound to the exterior surface of inner
- binds to active site and compete with substrate mitochondrial membranes of muscles, brain and liver
- migrates cathodal to CK-MM
non competitive - Indicator of severe illness
- binds at allosteric site
- changes the shape or spatial configuration of active site Macro CK
- migrates midway CK-MM and CK-MB
uncompetitive - comprised of CK-BB complexed with IgG,
- inhibitor binds to the ES complex - Its presence causes elevation of CK BB
- CK-Mi – to be detected must have an extensive tissue
MEASURE OF ENZYME ACTIVITY damage causing breakdown of mitochondrion and cell
- catalytic activity is measured and not the concentration wall. Does not correlate with any specific disease
- increase in product formation (fixed time method)
- decrease in substrate concentration CK MB
- decrease in co enzyme concentration - Following MI
- increase in concentration of altered co enzyme - Rise: 4-8 hours
- done at zero order kinetics - Peak: 24-48 hours
- Return to normal levels: 48-72 hours
all measurement enzyme done at zero order kinetics
METHODS OF ISOENZYME MEASUREMENT
METHODS OF MEASURING ENZYME ACTIVITY Electrophoresis
Fixed Time - may visualize adenylate kinase
- end point - reference method
- measures only 1 ab Ion exchange chromatography
- more sensitive and precise
Continuous monitoring or kinetic assay Immunoassays (RIA and immunoinhibition)
- kinetic or rate method - Abs against both M and B subunits
- measures more than 1 ab - measure ENZ concentration rather than activity

CREATINE KINASE CK METHODS

- associated with ATP regeneration in contractile or transport Tanzer-Gilvary (FORWARD)
system - coupled with PK-LD- NADH system
- optimum pH of 9.0
creatine + atp —> creatine phosphate + ADP Creatinine + ATP CK creatine phosphate + ADP
ADP + phosphoenolpyruvate PK pyruvate + ATP
- tissue sources: Pyruvate + NADH + H+ LD lactate + NAD +
- skeletal muscle, heart muscle, brain tissue
- physiologic function: occurs in the muscle cells Oliver Rosalki (REVERSE)
- for every contraction cycle of muscle results in the use of - coupled with HK-G6PD-NADH system
creatine phosphate with the production of ATP - most commonly performed method
- diagnostic significance: - optimum pH of 6.0
- sensitive indicator of AMI, muscular dystrophy Creatine phosphate + ADP CK creatine + ATP
(Duchenne type) ATP + glucose HK ADP + G-6-P
- occasionally seen in CNS disorder such as seizures, G-6-P + NADP + G-6-PD 6-phosphogluconate + NADPH

nerve degeneration
- total serum CK levels: early diagnostic tool to identify SOURCES OF ERROR
Vibrio vulnificus infection - Hemolysis
- three isoenzymes: - Instability of CK
- CK-BB: brain type - Exposure to light
- CK-MB: hybrid type Reference Range
- CK-MM: muscle type Total CK
- CK-BB:CK - 1 Male: 46-171 U/L
- migrates fastest towards anode Female: 34-145 U/L
- CK-MB: CK – 2 CK MB
- CK-MM:CK – 3 <5% of total CK
- slowest mobility
CK – occurs as a dimer consisting of two sub units that can be
separated readily into three molecular forms.
CK MM – major isoenzyme in normal sera, striated muscle
and skeletal muscle
CK MB – undetectable to trace ( less than 6% of the total ck
CK BB – small quantity in serum.. Most of the techniques
available cannot detect ck bb
- increased in carcinoma of various organs, untreated
prostatic carcinoma and other acenocarcinomas.
- THEREFORE CKBB CAN BE A USEFUL TUMOR MARKER
LACTATE DEHYDROGENASE (LD) ASPARTATE AMINOTRANSFERASE (AST)
- Catalyzes interconversion of lactic and pyruvic acids by - Involved in the transfer of an amino group between
transferring hydrogen using coenzyme NAD+ aspartate and a-keto acids
- Lactate + NAD Pyruvate + NADH + H - Uses pyridoxal phosphate as coenzyme
- Tissue sources: - Tissue sources
- heart, liver, skeletal muscle, kidney and erythrocytes - cardiac tissue, liver, skeletal muscle, kidney, pancreas,
- Diagnostic significance erythrocytes
- cardiac disease: AMI
- hepatic disease: hepatitis, cirrhosis DIAGNOSTIC SIGNIFICANCE
- skeletal muscle disease: muscle injury Increased
- renal disease: acute renal infarct - AMI
- hematologic disease: acute lymphoblastic leukemia, - congestive heart failure
hemolytic disorders, pernicious anemia - hepatocellular disorders
- Acute Myocardial Infarction (AMI) - skeletal muscle disorders
- LDH Levels - pulmonary embolism
- Rise : 12 to 24 hours - acute pancreatitis
- Peak : 48 to 72 hours Decreased
- Elevated: 10 days - uremia
- Consists of 5 major isoenzyme fractions
- LD-1: migrates most quickly toward anode, AST METHODS
- LD-5: migrates the slowest Karmen method (kinetic or rate reaction)
- LD-2: major isoENZ fraction - coupled enzymatic reaction change in absorbance at 340
- LD-1 > LD-2 : flipped pattern; suggestive of AMI nm
- LD-6 : Alcohol dehydrogenase - decreased in absorbance is measured
- optimal pH is 7.3-7.8
LD METHODS Reitman-Frankel (reaction with DNPH)
Wacker Method - ketoacids is reacted to 2,4-dinitrophenyl-hydrazine to form
- mixture of phenazine methosulfate and nitroblue ketoacid hydrazones in the presence of NaOH
tetrazolium reacts with NADH to produce a blue purple - product: intense brown color measured at 505 nm
color SOURCES OF ERROR
Wroblewski La Due Method - Hemolysis
- NADH serves as cosubstrate and is consumed during the - Substances that cause falsely elevated results:
course of reaction - bilirubin
- 3X faster than forward reaction - aceto-acetate and N-acetyl compounds
- p-aminophenol
LD ISOENZYMES - sulfathiozole
- Electrophoresis - isoniazid
- Immunoinhibition or chemical inhibition methods - ascorbic acid
- Differences in substrate affinity - Substances that may inhibit AST activity:
Assay for Enzyme activity - mercury
Lactate + NAD Pyruvate + NADH + H - cyanide
Forward reaction – pH 8.3 -8.9 - fluoride
Reverse reaction – pH 7.1 -7.14
ALANINE AMINOTRANSFERASE (ALT)
SOURCES OF ERROR - Catalyzes transfer of an amino group from alanine to a-
- Hemolysis ketoglutarate with formation of glutamate and pyruvate
- LS is unstable - Pyridoxal phosphate acts as coenzyme
- must be analyzed within 48 hours, stored at 25 degree - Tissue source
Celcius - liver and cardiac tissue
- LD-5 is the most labile isoENZ, analysis should be done
within 24 hours at DIAGNOSTIC SIGNIFICANCE
- REFERENCE RANGE: 125-220 U/L - Hepatic parenchymal disease like viral hepatitis,
obstructive jaundice, Reye’s syndrome
- AMI
- Muscular dystrophy
ALT METHODS
Karmen Method (coupled enzymatic method)
- Alanine + a-ketoglutarate ALT pyruvate + glutamate
- Pyruvate + NADH LD lactate + NAD +
- Indicator reaction: LD as the indicator ENZ catalyzes
reduction of pyruvate to lactate with simultaneous
oxidation of NADH monitored at 340 nm
SOURCES OF ERROR
- Relatively unaffected by hemolysis
- Substances that cause falsely elevated results:
- bilirubin, erythromycin, iproniazid, morphine -
- Substances that may inhibit ALT activity:
- heavy metals like mercurial diuretics
- REFERENCE RANGE: 6-37 U/L
ALKALINE PHOSPHATASE (ALP) ACID PHOSPHATASE
- Catalyze hydrolysis of various phosphomonoesters at an - Hydrolase that catalyzes hydrolysis of various
alkaline pH phosphomonoesters at an acidic pH (5.0)
- Optimal pH is 9.0-10.0 - Tissue source
- Requires Mg+2 and Mn+2 as activators - Prostate (richest source), bone, liver, spleen, kidney,
- Tissue sources RBC, platelets
- intestine, liver, bone, spleen, placenta, kidney
DIAGNOSTIC SIGNIFICANCE
ISOENZYMES - Prostatic carcinoma
- Liver: major liver band and fast liver band ( Hepatobiliary - Investigation of rape cases
obstruction) - Bone diseases like Paget’s disease and Gaucher’s disease
- Bone ( highest – Paget’s disease) - Platelet damage
- Placenta - Prostatic fraction – inhibited by tartrate
- Intestine
- Carcinoplacental isoenzyme ACP METHODS
- Regan and Nagao - Substrates – orthophosphoric monoesters; same with that
of ALP except for pH
ISOENZYME DIFFERENTIATION - Bodansky Method
- Electrophoresis - B-glycerophosphate or Na-glycerophosphate
- Heat stability (56 ˚C for 10 mins) - King-Armstrong or Gutman-Gutman Method
- if after heating, remaining activity is < 20%, the source of - phenylphosphate or disodium phenylphosphate
ALP elevation is the bone
- if after heating, remaining activity is >20%, the source of SOURCES OF ERROR
ALP elevation is the liver - Delay in separation of serum from red cells (ACP from RC
- Chemical inhibition and platelet may have leaked)
- Phenylalanine inhibits intestinal (75%) and placental - Instability of ACP
(80%) ALP - Hemolysis
- Regan isoenzyme – most heat stable ( resist denaturation - REFERENCE RANGE: 0-3.5 ng/mL
at 65⁰C for 30min
- inhibited by phenylalanine GAMMA GLUTAMYL TRANSFERASE (GGT)
- Nagao – variant of Regan - Involved in the transfer of the gamma glutamyl from
- inhibited by L-leucine gamma glutamyl peptides to amino acids, H2O2 and other
- Seen in metastatic CA of pleural surfaces small peptides
- Adenocarcinoma of the pancreas and bile duct. - Involved in peptide and CHON synthesis, regulation of
tissue glutathione levels, transport of amino acids across
DIAGNOSTIC SIGNIFICANCE cell membrane
- Bone disorders: Paget’s disease, osteomalacia, osteogenic - Tissue source
sarcoma - tissues of kidney, brain, prostate, pancreas, liver
- Hepatobiliary disorders: more on obstructive conditions
than in hepatocellular disorders like biliary tract DIAGNOSTIC SIGNIFICANCE
obstruction - Hepatobiliary disorders
- Intestinal ALP: disease of the digestive tract and in patients - Patients taking enzyme inducing drugs like warfarin,
undergoing chronic hemodialysis phenobarbital, phenytoin
- Alcoholism
ALP METHODS - Acute pancreatitis and diabetes mellitus
- Bodansky method
- substrate: B-glycerophosphate or Na-glycerophosphate GGT METHODS
- King-Armstrong or Gutman-Gutman Method - Szasz Method
- substrate: phenylphosphate or disodium - Substrate: L-Y-glutamyl-p-nitroanilide
phenylphosphate - Indicator reaction: production of p-nitroanilide or 5-
amino-2-nitrobenzoate
SOURCES OF ERROR - REFERENCE RANGE
- Hemolysis - Male: 6-45 U/L
- Delayed analysis - Female: 5-30 U/L
- Diet may elevate the level in group B and O individuals
who are secretors
- High fat meal
- REFERENCE RANGE: 30-90 U/L
AMYLASE (AMS) GLUCOSE-6-PHOSPHATE DEHYDROGENASE
- Hydrolase enzyme (G-6-P-D)
- Catalyzes breakdown of starch and glycogen with - Oxidoreductase that catalyzes oxidation of glucose-6-
products glucose, maltose, dextrin phosphate to 6-phosphogluconate or the corresponding
- Smallest enzyme and can be filtered by glomerulus lactone
- Tissue source - Tissue source
- skeletal muscle, small intestine, fallopian tubes and - adrenal cortex, spleen, thymus, lymph nodes, RBC
urine
DIAGNOSTIC SIGNIFICANCE
DIAGNOSTIC SIGNIFICANCE - To maintain NADPH in reduced form
- Acute pancreatitis - NADPH: required to regenerate Sulfhydryl containing
- Salivary gland lesion like mumps and parotitis proteins like glutathione
- Intraabdominal diseases - Glutathione protects Hb from oxidation
- Renal insufficiency - Increased level in myocardial infarction, megaloblastic
- Diabetic ketoacidosis anemia
- Macroamylasemia
- AMS combines with Ig to form a large complex that is G-6-PD METHOD
not filterable by the glomerulus - G-6-P + NADP G-6-PD 6-phosphogluconate + NADPH + H+
- Red cell hemolysate is used to assay for enzyme
AMS METHODS deficiency
- Amyloclastic - Serum is used to assay enzyme elevations
- Measures the disappearance of starch substrate - REFERENCE RANGE: 10-15 U/g hemoglobin
- Saccharogenic
- Measures the appearance of the product
- Chromogenic
- Measures the increasing color from production of
product coupled with chromogenic dye
- Continuous monitoring
- Coupling of several enzyme systems to monitor
amylase activity

SOURCES OF ERROR
- Acute pancreatitis with hyperlipemia (TG inhibits AMS)
- Morphine and other opiates may falsely elevate AMS
- Saliva contamination
- Oxalates and citrates may falsely decrease the level of
AMS
- REFERENCE RANGE
- Serum: 25-130 U/L
- Urine: 1-15 U/hour

LIPASE (LPS)
- Hydrolyzes the ester linkages of fats to produce alcohols
and fatty acids
- Catalyzes the partial hydrolysis of dietary TG in intestine to
the 2-monoglyceride intermediate, with the production of
long chain fatty acids
- Tissue source
- pancreas, stomach, intestine
DIAGNOSTIC SIGNIFICANCE
- More specific for pancreatic disorder than AMS
- Used to differentiate the cause of AMS elevation, whether
pancreatic disease or salivary gland involvement
- Intraabdominal conditions
LPS METHODS
- Turbidimetric enzyme reaction
- Simpler and more rapid
- Titration: Cherry Crandall method
- Classic method
- Uses olive oil substrate
- Measures the liberated fatty acids by titration after 24-
hour incubation

SOURCES OF ERROR
- Hemolysis should be avoided because hemoglobin
inhibits activity of serum LPS
- REFERENCE RANGE: 0-1.0 U/mL