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1 ENZYMOLOGY

2 ENZYMES
• ______________
• Catalyze biochemical reactions without altering the equilibrium point of the reaction
or being consumed or changed in composition

3 DEINITION OF TERMS
• ______________
• Where the substance on which the enzyme acts (substrate)
• ______________
• Bind regulator molecules
• ______________
• Different forms of enzymes based on electrophoretic mobility, solubility, or resistance
to inactivation
• ______________
• Results when an enzyme is subject to posttranslational modifications

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• ______________
• Nonprotein molecule that may be necessary for enzyme activity
• Inorganic cofactors
• ______________
• ______________
• Coenzyme/organic cofactors
• ______________
• Prosthetic group
• Enzyme portion (______________) + coenzyme (______________)
• Forms a complete and active system, a ______________
• ______________ or ______________
• Inactive form of enzyme
5 ENZYME CLASSIFICATION AND
NOMENCLATURE
• Systematic name
• Defining the substrate acted on
• The reaction catalyzed
• The name of any coenzyme involved in the reaction
• Recommended name
• When systematic name is too long
• EC numerical code containing four digits separated by decimal points
6 FIRST DIGIT
• Six classes
1. ______________
2. ______________
3. ______________
4. ______________
5. ______________
6. ______________

7 FACTORS THAT INFLUENCE ENZYMATIC REACTIONS

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7 FACTORS THAT INFLUENCE ENZYMATIC REACTIONS

8 SUBSTRATE CONCENTRATION
• __________________________
• The substrate readily binds to free enzyme at a low-substrate concentration
• ______________
• The substrate readily binds to free ________ at a low-substrate concentration
• The reaction rate is directly proportional to substrate concentration
• ______________
• Free enzyme combines with excess free _________
• The reaction rate depends only on enzyme concentration

9 MICHAELIS-MENTEN CONSTANT (KM)

• Derived from the theory of Michaelis and Menten
• An expression of the relationship between the velocity of an enzymatic reaction and
substrate concentration

10 PH
• Changes in pH may _________ an enzyme or influence its ______________
• Most physiologic enzymatic reactions occur in the pH range of 7.0 to 8.0

11 TEMPERATURE
• Increasing temperature usually increases the rate of a chemical reaction
12 COFACTORS
• Nonprotein entities that must bind to particular enzymes before a reaction occurs
• Inorganic cofactors
• ______________ (Ca2+,Fe2+, Mg2+,Mn2+, Zn2+, and K+)
• ______________ (Br- and Cl–)
• Organic cofactors
• Nucleotide phosphates
• ______________

•
13 INHIBITORS
• Interferes with the reaction
• ______________ inhibitors
• Physically bind to the ______________ of an enzyme
• ______________ inhibitor
• Binds an enzyme at a place ______________ the active site
• ______________ inhibition
• Inhibitor binds to the ES complex
• The enzyme–substrate–inhibitor
• complex does not yield product
14 MEASUREMENT OF ENZYME ACTIVITY
• Activity is related to concentration
• Measures increase in product concentration photometrically
• Always performed ______________
15 2 GENERAL METHODS IN MEASURING EA
• ______________
• The reactants are combined, the reaction proceeds for a designated time, the reaction
is stopped and a measurement is made of the amount of reaction that has occurred.
• ^ rxn = ^ enzyme
• ____________________________
• Multiple measurements, usually of absorbance change, are made during the reaction,

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• ____________________________
• Multiple measurements, usually of absorbance change, are made during the reaction,
either at _______ ________(usually every 30 or 60 seconds) or continuously by a
continuous-recording spectrophotometer
• Advantageous over fixed-time methods
16 CALCULATION OF ENZYME ACTIVITY
• ______________s
• The units used to report enzyme levels
• ______________(IU)
• The amount of enzyme that will catalyze the reaction of 1 mol of substrate per minute
• When enzymes are quantitated by measuring the increase or decrease of NADH at 340
nm, the molar absorptivity (6.22 103 mol/L) of NADH is used to calculate enzyme activity.

17 ENZYMES AS REAGENTS
• Enzymes may be used as reagents to measure many nonenzymatic constituents in serum
• ______________ enzymes
• Chemically bonded to adsorbents, such as agarose or certain types of cellulose, by
azide groups, diazo, and triazine

18 CREATINE KINASE

• Molecular weight = approximately ______________

• Predominant physiologic function occurs in ______________
• Storage of high-energy creatine phosphate
• Highest activities found in ______________ ______________ ______________
• Smaller quantities in bladder, placenta, gastrointestinal tract, thyroid, uterus, kidney,
lung, prostate, spleen, liver, and pancreas

19 DIAGNOSTIC SIGNIFICANCE
• Frequently elevated in disorders ______________ ______________
• Sensitive indicator of acute myocardial infarction (AMI) and muscular dystrophy
(_________________________)
• Duchenne type = 50 to 100 times the upper limit of normal (ULN)
• Vary with muscle mass

20 THREE ISOENZYMES
• CK-BB (______________ type)
• CK1
• CK-MB (______________ type)
• CK 2
• CK-MM (______________ type)
• CK 3
21 METHODS OF MEASUREMENT
• ______________
• Reference method
• Allowing visualization of adenylate kinase (AK) (hemolyzed)
• ____________________________
• ______________
• ______________

22 ASSAY ENZYME ACTIVITY

• Change in absorbance at ___________is determined

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22 ASSAY ENZYME ACTIVITY

• Change in absorbance at ___________is determined
• Forward reaction is coupled with the pyruvate kinase–LDH–NADH
• pH _________
•

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• Reverse reaction is coupled with the hexokinase–glucose-6-phosphate dehydrogenase–
NADP system
• pH ____

24 SOURCE OF ERROR
• AK reacts with ADP to produce ATP
• Causing falsely elevated CK levels
• CK is ______________
• Activity can be restored after storage in the dark at ________________________ when the
assay is conducted using a sulfhydryl activator
25 REFERENCE RANGE
• Total CK:
Male, ______________
Female, ______________
• CK-MB: ______________

26 LACTATE DEHYDROGENASE

• Catalyzes the interconversion of _________________________

• High activities are found in the __________________________________________
• Lesser amounts are found in the lung, smooth muscle, and brain

27 DIAGNOSTIC SIGNIFICANCE
• LDH is elevated in a variety of disorders
• Highest levels of total LDH = ______________ and ______________
• In AMI,
• Rise ______________
• Peak ______________
• May ______________

28 SOURCE OF ERROR
• Any degree of hemolysis should render a sample unacceptable for analysis
• LDH activity is unstable in serum
• If the sample cannot be analyzed immediately, it should be stored at ______________
______________

29 REFERENCE RANGE
• LDH, ______________ (37°C)
30 ASPARTATE AMINOTRANSFERASE
• Transferases
• Commonly referred to as a ______________
• Serum ________________________(SGOT, or GOT)
•
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•
31 TISSUE SOURCE
• The highest concentrations are found in cardiac tissue, liver, and skeletal muscle
• Smaller amounts found in the kidney, pancreas, and erythrocytes.

32 DIAGNOSTIC SIGNIFICANCE
• In AMI
• Rise within ______________
• Peak at ______________
• Return to normal ______________
• Elevations are frequently seen in ______________
• Highest in ______________ disorders
• In ______________, levels may reach ______________ ULN

33 ASSAY FOR ENZYME ACTIVITY

• AST are generally based on the principle of the ______________
• Coupled enzymatic reaction using ______________(MD) as the indicator reaction and
monitors the changein absorbance at ______________ continuously as NADH is oxidized
to NAD+
• pH ______________

34 SOURCE OF ERROR
• ______________ should be avoided because it can dramatically ______________ serum AST
concentration
• Stable in serum for ______________ at refrigerated temperature
• Reference Range
• ______________ U/L (37°C)
35 ALANINE AMINOTRANSFERASE

• Catalyzes the transfer of an amino group from alanine to a-ketoglutarate with the
formation of glutamate and pyruvate
• _________________________(SGPT, or GPT)
•
36 TISSUE SOURCE
• High concentrations in the ______________
• Considered the more ______________ enzyme of the transferases

37 DIAGNOSTIC SIGNIFICANCE
• Mainly to evaluation of ______________
• Remains normal in AMI
•

38 ASSAY FOR ENZYME ACTIVITY

• Coupled enzymatic reaction using ______________ as the indicator enzyme
• Change in absorbance at 340 nm measured
39 SOURCE OF ERROR
• ALT is stable for 3 to 4 days at 4°C. It is relatively unaffected by hemolysis.

• Reference Range

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• Reference Range
• ______________ (37°C)

40 ALKALINE PHOSPHATASE

• Catalyze the hydrolysis of various phosphomonoesters at an ______________ pH

• Functions to liberate inorganic phosphate from an organic phosphate ester
• Optimal pH for the reaction is ______________
• The enzyme requires ______________ as an activator

41 TISSUE SOURCE
• Present on cell surfaces in most human tissue
• Highest concentrations are found in the intestine, liver, bone, spleen, placenta, and
kidney
• In the liver, the enzyme is located on both sinusoidal and bile canalicular membranes
• Activity in bone is confined to the ______________
• Production of bone matrix

42 DIAGNOSTIC SIGNIFICANCE
• Evaluation of hepatobiliary and bone disorders
• ______________ > ______________
• Highest elevations in ______________(______________)
43 ISOENZYMES
• ______________
• ______________
• ______________
• ______________

44 ASSAY FOR ENZYME ACTIVITY

• Calculation of ALP activity based on the molar absorptivity of ______________
• p-Nitrophenylphosphate (______________) is hydrolyzed to p-nitrophenol (______________)
• Increase in absorbance at ______________
•
45 SOURCE OF ERROR
• Hemolysis may cause slight elevations
•
• Reference Range
• ______________ (30°C)
46 ACID PHOSPHATASE
• Same with ALP
• Optimal pH of ______________
47 TISSUE SOURCE
• Found in the prostate, bone, liver, spleen, kidney, erythrocytes, and platelets
48 DIAGNOSTIC SIGNIFICANCE
• Historically, ACP measurement has been used as an aid in the detection of ______________,
particularly metastatic carcinoma of the ______________
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particularly metastatic carcinoma of the ______________

49 SOURCE OF ERRROR
• Serum should be separated from the red cells as soon as the blood has clotted
• CP is stable for ____________________________.
•
• Reference Range
• Prostatic ACP ______________
50 Y-GLUTAMYLTRANSFERASE
• involved in:
• ______________ synthesis
• Regulation of tissue ______________ levels
• Transport of ______________ across cell membranes

51 TISSUE SOURCE
• Found primarily in tissue of the ________________________________________
• Clinical applications are focused on ______________ and ______________ disorders
52 DIAGNOSTIC SIGNIFICANCE
• Elevated in virtually all ______________
•
53 ASSAY FOR ENZYME ACTIVITY
• p-nitroaniline at ______________ nm

54 SOURCE OF ERROR
• Stable for 1 week at 4°C
• No GGT in rbc
•
• Reference Range
• GGT: male______U/L (37°C); female, _____ U/L (37°C)
55 AMYLASE
• Catalyze the breakdown of ______________

56 TISSUE SOURCE
• ______________ of the ______________ and the ______________
• Lesser concentrations are found in skeletal muscle and the small intestine and fallopian
tubes
• AMS is the smallest enzyme, with a molecular weight of ______________
57 DIAGNOSTIC SIGNIFICANCE
• Diagnosis of ______________
• In ______________, serum AMS levels
• Rise at _________________________
• Peak at _____
• Return to normal levels within ________
• Range from ______________ Somogyi units per dL (2.55 ULN)
• Salivary gland lesions, such as mumps and Parotitis, and other intra-abdominal diseases,
such as perforated peptic ulcer, intestinal obstruction, cholecystitis, ruptured ectopic
pregnancy, mesenteric infarction, and acute appendicitis
58 ISOENZYMES
• P isoamylase = ______________
• (P1, P2, P3)
• S isoamylase = ______________
• (S1, S2, S3)
59 ASSAY FOR ENZYME ACTIVITY
• optimal pH is ____
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• optimal pH is ____
60 SOURCE OF ERROR
• Little loss of activity occurs at room temperature for _________________________
• ______________ and other opiates = falsely elevated serum AMS levels
• Reference Range
• Serum, ________; urine, ________
• Conversion factor is _______
61 LIPASE
• Hydrolyzes the ______________ to produce ______________ and ______________
62 TISSUE SOURCE
• Found primarily in the ______________
• Also present in the stomach and small intestine

63 DIAGNOSTIC SIGNIFICANCE
• Confined almost exclusively to the diagnosis of ______________
• More specific for pancreatic disorders than AMS
• LPS elevations persist for approximately 5 days in acute pancreatitis
64 ASSAY FOR ENZYME ACTIVITY
• __________________used an olive oil substrate
65 SOURCE OF ERROR
• Negligible loss in activity at room temperature for ___________________________
• Hemoglobin inhibits the activity of serum LPS, causing falsely low values
• Reference Range
• ______________
66 GLUCOSE-6-PHOSPHATE DEHYDROGENASE
• Catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate or the
corresponding lactone
• Tissue Source
• Adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, and
erythrocytes
• Little activity is found in normal serum
67 DIAGNOSTIC SIGNIFICANCE
• Functions to maintain NADPH in reduced form
• Regenerate sulfhydryl-containing proteins(glutathione)
• Protects hemoglobin from oxidation by agents that may be present in the cell
• Reference Range
• ______________
68 MACROENZYMES
• Serum enzymes that can be bound to either an ______________ (macroenzyme type 1) or a
________________________(macroenzyme type 2)
• Found in patients who have an unexplained persistent increase of enzyme
concentrations in serum
69 DRUG-METABOLIZING ENZYMES
• Function primarily to transform xenobiotics into inactive, water-soluble compounds for
excretion through the kidneys
• ______________
• Catalyze addition or removal of functional groups through hydroxylation, oxidation,
dealkylation, dehydrogenation, reduction, deamination, and desulfuration reactions
• Often mediated by _______________ (_______) enzymes
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• ______________
• Xenobiotics transformed into more polar compounds through enzyme-mediated

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• ______________
• Xenobiotics transformed into more polar compounds through enzyme-mediated
conjugation reactions
• conjugated with glucuronide (UDP-glucuronyltransferase 1A1 [UGT1A1]), acetate (N-
acetyltransferase [NAT]), glutathione (glutathioneS-transferase [GST]), sulfate
(sulfotransferase), and methionine groups