Chem Enzymes Flashcards
1
Q
Biologically synthesize proteins that catalyze biochemical reactions without altering the equilibrium point or being consumed in the process
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Enzymes
2
Q
What are enzymes a marker for?
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Cellular injury or degrading cells, tissue damage
3
Q
Primary protein structure
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Linear amino acid sequence
4
Q
Secondary protein structure
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Steric arrangement of polypeptide chains
5
Q
Tertiary protein structure
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Folded arrangement resulting in structural cavities
6
Q
Quarternary protein structure
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Interaction of multiple units, enhances the control of an enzyme or catalytic ability of a subunit
7
Q
Isoenzymes
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Multiple forms of an enzyme catalyzing the same biochemical reaction
8
Q
Isoforms
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Multiple forms of isoenzymes that result from enzymatic modification of the parent form after its release from the tissues
9
Q
Cofactor
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Nonprotein molecule necessary for enzyme activity
10
Q
Activator
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Inorganic cofactor such as chlorine or magnesium ions, small molecules
11
Q
Coenzyme
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Organic factor such as NAD, NADP, vitamin derivatives; large molecules
12
Q
Prosthetic group
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Coenzyme tightly bound to an enzyme
13
Q
Apoenzyme
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Protein portion of enzyme without co-factor which shows little or no enzymatic activity
14
Q
Holoenzyme
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Apoenzyme + coenzyme = haloenzyme; complete active system
15
Q
Proenzyme (zymogen)
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Inactive enzyme later altered to become active; digestive enzymes like trypsinogen to trypsin when needed, prevents auto digestion
16
Q
Systematic name
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1. Name of the substrate acted on
2. -ase: Indicates the type of reaction catalyzed by all enzymes in the group
17
Q
E.C. Nomenclature
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Systematic name, EC code number, practical name/trivial name, standard abbreviation
18
Q
E.C. code number
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- Enzyme class
- Subclass
- Sub-subclass
- Serial number
Lactate dehydrogenase (LD)
1.1.1.27
19
Q
Enzyme classes
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1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases
20
Q
Oxidoreductase
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Catalyze oxidation–reduction reaction between two substrates; OIL RIG
21
Q
Transferases
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Catalyze the transfer of a group other than hydrogen from one substance to another
22
Q
Hydrolases
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Catalyze hydrosisis of various bonds; splitting of water
23
Q
Lyases
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Catalyze removal of groups from substrates without hydrolysis; splitting without water, product contains double bonds
24
Q
Isomerases
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Catalyze the intra-conversion of geometric, optical, or positional isomers
25
Ligases
Catalyze the coupling of two compounds by utilizing the energy of an ATP or other nucleoside triphosphate
26
Slow rate in enzyme kinetics
Insufficient kinetic energy to drive reaction to form products
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Activation energy
Excess energy, energy required to raise all molecules in one mole of compound to transition state at peak of energy barrier
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Enzymes affect on activation energy
Enzymes lower activation energy that the substrates must reach for the reaction to occur
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Enzyme-substrate complex
Can revert back to original molecules or complete the reaction and form product and regenerated enzyme
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Enzyme kinetic specificity
Absolute: only one substrate catalyzes only one reaction
Group: all substrates containing a particular chemical group
Bond: specific bonds within substrates
Stereoisomeric: only a single optical isomer of a certain compound
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Theories of substrate binding by enzymes
1. Lock and key
| 2. Induced fit
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Factors that influence enzymatic reactions
1. substrate concentration
2. Enzyme concentration
3. pH
4. Temperature
5. Presence of inhibitors, activators, coenzymes, and prosthetic groups
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Michaelis-Menten curve
Relationship between reaction velocity and substrate concentration in an enzymatic reaction
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Km
Substrate concentration at which the reaction velocity is half of the maximum level
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Michaelis-Menten Equation
V = Vmax * [S] / Km + [S]
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Lineweaver-Burk transformation
Inverse of M-M equation
Reciprocal of y-intercept 1/Vmax
Negative reciprocal of x interval -1/Km
Slope = Km/Vmax
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First-order kinetics
Rate dependent on substrate concentration (excess enzyme)
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Zero-order kinetics
Excess substrate present and all available sites on the enzyme are saturated with substrate and reaction velocity reaches maximum
-rate depends on enzyme concentration
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Zero order kinetics factors
Entire enzyme is bound to substrate and a much higher rate of reaction is obtained
- rate of enzyme catalysis is constant
- independent of substrate concentration
- dependent on enzyme activity
- linear with time
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Enzyme concentration
Substrate concentration exceeds enzyme concentration
-velocity/reaction is proportional to enzyme concentration
Higher enzyme level, faster reaction
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pH
Denature or influence ionic state
-structural changes, change in the charge
Most occur at pH 7-8, pepsin: pH 1-2,
ALKP: pH 10
In lab, pH controlled by buffer solutions
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Temperature
Ideal 37 C
Higher may denature enzyme
Repeated thawing/refreezing denatures enzymes
Storage conditions
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Presence of inhibitors, activators, coenzymes, and prosthetic groups
Cofactors
Excess so that rate not dependent on cofactor concentration
Inhibitors
Competitive, noncompetitive,
Uncompetitive
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Competitive inhibition
Substrate and the inhibitor compete for the active site of the enzyme, Vmax is the same and aparent Km increased
Graph lines cross
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Apparent Km
Effective affinity for the substrate is reduced
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Noncompetitive inhibition
Bind to the site other than the active site, called an allosteric site
Vmax is decreased
Km is unchanged
Graph starts at same point but one has a steeper slope
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Uncompetitive inhibition
Inhibitor binds only to the enzyme-substrate complex and not to the free enzyme
Vmax is decreased
Apparent Km is decreased
Graph lines are parallel
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Reaction phases
Lag period, log period/linear or zero-order, nonlinear period/plateau
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Measuring enzyme activity
1. Increased product concentration
2. Decreased substrate concentration
3. Increased/decreased coenzyme concentration
Enzyme concentrations are performed on zero order, with substrates and coenzymes added in excess
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Coupled enzyme assays
1. Coenzyme
2. Auxiliary enzyme
3. Indicator enzyme
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Coenzyme
Not involved in main reaction is added in excess
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Auxiliary enzyme
Intermediate enzyme upon which product of reaction becomes substrate for auxiliary reaction
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Indicator enzyme
Enzyme which catalyze its final reaction where there is in absorbance change
NAD to NADH
Increase in abs
54
Two General methods used to measure the extent of an enzymatic reaction
Fixed-time and continuous monitoring (kinetic)
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Fixed-time method
Reactants combine and allowed to react for designated amount of time, reaction is stopped usually by inactivating the enzyme; The larger the reaction the more enzyme present
56
Continuous-monitoring method
Multiple measurements usually of an absorbance change are made during the reaction at specific time intervals, measured during log/linear phase
57
Enzyme calculations
1. Quantitated relative to their activity
2.  rate of reaction is proportional to enzyme concentration
3. International unit, the amount of enzyme that will catalyze one micromole of substrate per minute
58
Immunoassay mass measurements of enzymes
Concentration reported rather than activity, may overestimate active enzymes as a result of possible cross-reactivity
CKMB is only mass enzyme
Electrophoresis can be used to quantify enzymes
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Measure non-enzymatic constituents
1. Certain molecules measured with enzymes
2. Specificity of enzyme gives true level of analyte
3. Methods to quantify analytes that are substrates for the corresponding enzyme
60
Immobilized enzymes
1. Chemically bonded to gel or cellulose
2. Convenient for continuous flow
3. More stable than enzymes in solution
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Immunoassays
Used as reagents incompetitive and noncompetitive immunoassays, function as an indicator that reflects either presence or absence of the analyte
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Plasma specific enzymes
Thrombin, factor XII, factor X
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Secreted enzymes
Lipase, amylase, pepsinogen
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Cellular enzymes
LDH, ALT, AST, ALP
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Factors affecting serum levels
1. Leakage from cells, anything that can cause cell death
2. Altered enzyme production rate
3. Enzyme induction
4. Proliferation
5. Plasma half-lives
6. Renal clearance 
66
Creatine Kinase (CK) (CPK)
1. Striated muscle, heart, brain, some from smooth muscle (mainly GI)
2. Hemolysis artifactual - AK
3. Neonatal period or first few days after child birth will be raised
- Marked increase in shock or circulatory failure, myocardial infarction, MS
- Moderate increase in muscle injury, drugs, after surgery, physical exertion, seizures, hypothyroidism, alcoholism
- Plasma CK is raised in all forms of muscular dystrophy but not neurogenic muscular diseases
4. Normal isoenzymes: CK-BB, CK-MB (1,2), CK-MM (1,2,3), atypical: macro CK and mitochondrial CK
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CKMB relative index %
(CK-MB ng/mL / total CK IU/L ) x 100 =RI%
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Calculate % of CK-MB
(CK-MV IU/L / CK IU/L) x 100 = %
69
Measurement of CKMB
Electrophoresis
Immunoinhibition
*Sandwich immunoassay - best, antibodies are more specific for isoenzyme, less time consuming
Ion-exchange chromatography
70
Lactate dehydrogenase (LD) (LDH)
1. Widely distributed- Heart, liver, skeletal muscle, kidney, brain, you were three sites, present in cytosol of all human cells
2. Something somewhere is damaged “smoke signal”
3. Hemolysis is artifactual
4. Marked elevation - pernicious anemia and hemolytic disorders, circulatory failure with shock and hypoxia, rejection of renal transplant
- moderate increase, viral hepatitis and cirrhosis, malignancy, skeletal muscle disease, MI, PE, mono
- mild, thalassemia, myelofibrosis, hemolytic anemia
71
LDH subunits
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LD1 - HHHH mainly heart and RBC
LD2 - HHHM mainly heart and RBC
LD3 - HHMM lung
LD4 - HMMM liver, muscle
LD5 - MMMM liver, muscle
a-hydroxybutyrate: substrate specific for H subunit used to separate LD1 from other isoenzymes
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72
Aminotransferases (transaminases)
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Aspartate aminotransferase (AST)
Alanine aminotransferase (ALT)
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73
Aspartate aminotransferase (AST) (GOT) (SGOT)
1. Highest concentration found in cardiac tissue, liver, and skeletal muscle
2. Hemolysis is artifactual
3. Physiological during neonatal period
4. Marked increase, heptocellular disorders, Circulatory failure with shop and hypoxia, acute or viral hepatitis, AMI
- moderately raised, cirrhosis, mono, cholestatic jaundice, malignant infiltration, skeletal muscle disease…
74
Alanine aminotransferase (ALT) (GPT) (SGPT)
1. Highest concentration in the liver
2. More liver specific that AST,
- Normal ALT with increased AST indicates cardiac origin
- Higher elevations are found in hepatocellular disorders
3. Marked elevation, circulatory failure with shock and hypoxia, acute viral or toxic hepatitis
- moderately raised, cirrhosis, mono, liver congestion, cholestatic jaundice
75
Alkaline phosphatase (ALP)
1. Bone, liver, intestinal, placental
2. Preterm infants, children until puberty, last trimester pregnancy, bone healing after fracture
- Bone disease: osteomalacia and rickets, pagets disease, bone cancer, etc
- liver disease: intra and extrahepatic cholestasis, space occupying lesions, tumor, granulomas, and other hepatic infiltration
- malignancy: bone or liver cancer
- other: Hodgkin’s, CHF, ulcerative colitis, regional enteritis, peritonitis
3. Measurement or isoenzymes
- heat fractionation measure, heat, measure again
- electrophoresis
- chemical inhibition
76
Calculation of % heat stable alkaline phosphatase
Retest ALP / initial ALP X 100 = % isoenzyme
77
Gamma-glutamyltransferase (GT) (GGT) (gamma GT) (GGTP)
1. kidney, brain, prostate, pancreas, liver
- Sensitive indicator of hepatobiliary disease
- Useful in diagnosis of obstructive jaundice and chronic alcoholic liver disease
- Important in absorption of amino acids from intestinal lumen
2. Marked increase, alcoholic hepatitis or gross alcohol abuse, cholestatic liver disease, post-hepatic obstruction
- moderate elevation, viral hepatitis, fatty liver, drug effects
- normal, bone disease, prostate carcinoma
78
GGT is a
Transpeptidase, catalyzes the transfer of the gamma glutamyl group 
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Two important liver cells
Hepatic cells and kupffer cells
80
Four common liver enzymes
AST, ALT, ALP, GGT,
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More hepatocellular enzymes
AST and ALT
82
More liver obstruction enzymes
GGT and ALP
83
Amylase - smallest enzyme
1. Acinar cells in pancreas (P1, P2, P3), saliva (S1, S2, S3)
Increased P3: Pancreatitis and in renal failure
2. Marked elevation, acute pancreatitis, glomerular impairment, severe diabetic ketoacidosis, Perforated peptic ulcer/pseudocyst
-moderate elevation, Acute abdominal disorders, call bladder infection, intestinal obstruction, abdominal surgery/trauma, ruptured ectopic pregnancy, salivary gland disorders, etc.
84
Urine amylase
Only enzyme that can pass through the glomerioli of the kidneys
-if lipemic serum do urine analyze
-lipemia found in diabetes mellitus, nephrotic syndrome, pancreatitis
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Macroamylase
Cannot go through kidneys, stay in serum
-immunoglobulin binds to amylase so that it cannot be excreted in urine, appearance of false pancreatitis, do urine to check
86
Pancreatic pseudocyst
If plasma enzyme levels fail to fall after attack of acute pancreatitis, may have leakage of fluid into lesser sack of pancreatic peritaneum
-urinary amylase levels are high differentiating between macroamylasemia
87
Measurement or amylase
- amyloclastic
- saccharogenic
- chromogenic (chromolytic)
- continuous-monitoring
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Lipase
1. Produce primarily by acinar cells of the pancreas, present in stomach and small intestine
2.  physiological role to hydrolyze long chain triglycerides in small intestine
3. More specific than amylase for pancreatic disorders, less sensitive
4. Increased in pancreatitis, pancreatic obstruction, Common bile duct obstruction, pancreatic abscess, pancreatic cancer
5. Lipase levels useful in differentiating a serum Annalise elevation due to pancreatic versus salivary involvement
89
Cholinesterases
Two: enzymes have the ability to hydrolyze acetylcholine
1. True cholinesterase (AChE)
- found in RBCs primarily
-repolarize nerves
2. Pseudocholinesterase (PChE)
-found in plasma/serum
-biological role is unknown

3. Succinyl-choline, muscle relaxant used in surgery, Hydrolysis by cholinesterase to remove from body
-if decreased cholinesterase activity, drug not destroyed rapidly enough and person may enter prolonged apnea
-Insecticide poisoning, both AChE and PChE inhibited by organic phosphates
4. Dibucaine or fluoride act as PChE inhibitors

90
Acid phosphatase (AP) (ACP)
1. Mainly prostate, also liver, spleen, kidney, RBC, PLT
2. Increased in prostate CA and Gauchers/pagers
PSA Better test for prostate, ACP not elevated until late in CA
3. Specimen handling, very unstable add Acid to preserve
4. Vaginal sample in rape battery - legal issues
91
ACP isoenzyme methods
- specific substrate
- chemical inhibition
Total ACP - ACP after tartrate inhibition = prostatic ACP
92
ACP sources of error
- serum should be separated from RBCs as soon as blood has clotted to prevent leakage of erythrocytes platelet ACP
- room temp, only good 1-2 hours without preservative
- hemolysis should be avoided
93
Prostate specific antigen (PSA)
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Single chain glycoprotein
Two major forms in the blood:
1. complexed with another molecule
2. Free
-useful as a tumor marker
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Glucose-6-phosphate dehydrogenase
1. Mainly RBCs, also adrenal cortex, spleen, lymph nodes, lactating mammary gland
- functions in production of NADPH
- can lead to formation of Hines bodies
2. Differs from normal routine in 2 ways
- Detect abnormal alleles to aid in the investigation of hemolytic anemia instead of detecting tissue damage
- Sample used is RBCs instead of serum or plasma
