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Laboratory Reviewer 2
Experiment 3: Factors That Affect Enzyme Activity
Enzymes
• conjugated proteins that catalyze biological reactions by lowering the energy of activation (ΔG)
• act on specific substrates & are regenerated after every reaction
Alkaline Phosphatase
• Enzyme used in the experiment extracted from rat intestines
• Class III: Hydrolase à non-specific metalloenzyme
• Activator: Magnesium Ions - from MgCl 2
• Optimum pH
o 9.80 – 10.00
o maintained by Glycine Buffer (Buffering range: 8.80 – 10.60)
• Optimum Temperature
o 37ºC
o maintained by water bath
• Substrate: Sodium B-Glycerophosphate
• Reaction Catalyzed:
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
• Dialysis of Alkaline Phosphatase
o Dialysis
§ separation of the naturally-bound Magnesium Ions from
the enzyme structure
§ uses a selectively permeable membrane (dialyzing bag)
with pores to allow the Small Magnesium ions to go out
of the dialyzing bag & allow the apoenzyme/inactive
enzyme to be trapped within the bag
§ must be done in a cold temperature solution for the
enzyme to be inactive during the dialysis
o Enzymes to be used per experiment
§ Undialyzed/Active Holoenzyme
• Effect of pH & Temperature
• To ensure a catalytically active enzyme that would ONLY be affected by the variables of
temperature and pH
§ Dialyzed/Inactive Apoenzyme
• Effect of Activators & Inhibitors
• The variable being tested in the effects of Activators and Inhibitors was the presence of
cofactors which would affect enzyme activity. We would not be able to observe the effect
of its absence/pressence if there are naturally-bound cofactors to the enzyme
• Dialysis would eliminate this problem by removing the naturally-bound cofactors to be able
to observe the true effects of activators & inhibitors
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
o pH (Bell-shaped curve)
§ Ascending Limb
• épH: éActivity
• during this time, we modify the amino acid charged groups toward
the proper functional enzyme folding pattern
§ Optimum pH
• pH at which enzyme is at its maximum activity
• optimum/proper/functional folding pattern of the enzyme
§ Descending Limb
• épH: Denaturation of enzyme – unfolding of the polypeptide chain
and loss of catalytic activity
§ pH Tested: 1.00, 7.00, 8.50, 10.50
§ pH was measured by pH meter
§ pH was altered by HCl (acids decrease pH) & NaOH (bases increase pH)
o Activators
§ Metal ion cofactors act as activators of catalysis by
• binding to substrate to orient them properly for reaction
• mediating oxidation-reduction reactions through reversible changes in the metal ions
oxidation state
• electrostatically stabilizing or shielding negative charges
§ Activators Tested: Mg+2 (from MgCl ), Ca+2 (from CaCl ), Fe+3 (from FeCl )
2 2 3
§ Magnesium Ions
• Known activator of Alkaline Phosphatase
• It localizes the pi electrons present in the double bond of PO4- to facilitate stabilization of
the negative charge.
• Stabilization/masking of the charge will allow the enzyme’s active site to nucleophillically
attack the substrate making catalysis possible
o Inhibitors
§ chemicals that bind to the enzyme and reduce the rate of enzymatic
reactions
§ inhibitors tested: chelating agents – bind to metal cofactors & make them
unavailable to the enzyme, hence inactivating it.
• Ethylene Diaminetetraacetic Acid
o better inhibitor as it has 4 COO- which could chelate 2Mg 2+
• Citrate
o has only 3 COO- which could chelate only 1Mg 2+
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
Where: S = substrate
E = enzyme
ES = enzyme-substrate complex
K , K and K = rate constants
1 =1 2
Michaelis-Menten Kinetics
• Describes how reaction velocity varies with substrate
concentration
• Graph
o V = Initial velocity
o Vmax = Maximum velocity, 100% of enzyme active
site is saturated
o ½ Vmax = 50% of enzyme active site is saturated
with substrate
o Km
§ Michaelis Constant (K-1+K2)/K1
§ (x =Km, y=1/2Vmax)
• get half the Vmax then trace to
the x-axis, that is your Km
§ reflects the affinity of the E for the S
• Small Km = High Affinity
o low [S] is needed to half-
saturate the enzyme
• Big Km = Low Affinity
o high [S] is needed to half-saturate the enzyme
o [S] = Substrate concentration
• Assumptions
o Assumption of Equilibrium
§ S binds reversibly with E
• initially P is small: E+SàES
• when P accumulates, reaction reverses ESàE+S
• Until equilibrium is reached catalyzed by the
enzyme
o Assumption of Briggs & Haldane
§ steady state assumption
§ rate of synthesis (E + S --> ES) = rate of degradation (ES --> E + P)
o Substrate Concentration is far > Enzyme concentration
§ enzyme will be limiting when the active sites are occupied
o The rate of the reaction is measured the moment S + E comes in contact: Initial Velocity
§ at the Initial Portion of the reaction
§ A lot of substrates available for conversion & no products are formed yet that may inhibit the
reaction (via negative feedback) so we are able to obtain maximum velocity at the start of the
reaction
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
• Reaction
o Pseudo-1 order
st
Lineweaver-Burke Plot
• graphical linearization of the Michaelis-Menten Kinetics to be able to determine Km and Vmax
• double reciprocal plot
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
Oxidation
Red
Brown
Semiquinone
*Read
absorbance
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
o Glycosylated Hemoglobin A1c (HbA1c)
§ excess blood glucose is non-enzymatically bound to amino acids of certain proteins like Hemoglobin
of RBC; more glucose in the blood stream, more Glycosylation of proteins would occur
§ measures the amount of Glucose (in %) that is attached to Hemoglobin of RBC
§ Since RBCs live for 120 days, Hb1Ac test shows the average blood sugar for the past several
months & not just the moment of post-fasting hence, it is used to monitor patient compliance to
DM treatments
§ Not affected by sudden fluctuations in blood glucose
§ Clinical Interpretations
• 6% 120 mg/dl (Normal)
• 8% 180 mg/dl (not too bad)
• 10% 240 mg/dl (not good)
• 13% 330 mg/dl (dangerous)
§ False Elevations
• Restricting carbohydrate intake may alter baseline results
• Illness may conceal a person’s actual glucose metabolism when healthy
• Exaggerated amounts of glucose may lead to erroneous results
GLUCOSE TOLERANCE CURVE
• Normal Patient
o Peak – 1 hour st
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
• Determination of Triglycerides
o Nature
§ TAGs/Neural Fats exists in the plasma
§ Formed by esterification of glycerol + 3 fatty acids
§ Major fat in the diet (>90% of dietary fat)
§ Main storage of fats in man; 95% of adipose tissue lipids
§ Found in plasma as part of lipoproteins in various concentration
§ Highest during absorption after meals (peak at about 4 to 6 hours after a meal, exogenous lipids
should be cleared from plasma before analysis)
o Assay
§ Hydrolysis of TAG to Glycerol + 3 Free Fatty Acids by Lipase
§ Glycerol is phosphorylated by Glycerol Kinase & ATP forming Glycerol-3-Phosphate
§ Glycerol-3-Phosphate is oxidized by Glycerol Phosphate Oxidase to Dihydroacetone Phosphate +
Hydrogen Peroxide (H O )2 2
o Specimen: Serum
o Reagents
§ PIPES buffer (pH 7.5), 4-chlorophenol, 4-aminoantipyrine, Magnesium ions, ATP, Lipases, Peroxidase,
Glycerol kinase, Glycerol-3-phosphate oxidase
o Computation
§ [TAG]mg/dL = (ASX/ASTD) x 200
§ [TAG]mmol/L = (ASX/ASTD) x 2.28
o Clinical Interpretation
§ SUSPECT IF ABOVE: 150mg/dL (1.71mmol/L)
§ ELEVATED RISK IF ABOVE: 200mg/dL (2.28mmol/L)
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
o Assay: 500nm
o Specimen: Serum
o Computation
§ [Cholesterol]mg/dL = A x 553
§ [Cholesterol]mmol/L = A x 14.3
o Clinical Interptretation: Hypercholesterolnemia
§ SUSPECT IF ABOVE: 220mg/dL (5.7mmol/L)
§ ELEVATED RISK IF ABOVE: 260mg/dL (6.7mmol/L)
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
Sources of Cholesterol
• Dietary absorption – exogenous cholesterol
• De Novo Biosynthesis – endogenous cholesterol
• Regulation
o Regulation of HMG-CoA Reductase
§ Competitive inhibition - statin drugs: êActivity
§ Feedback control - éDietary cholesterol, éBile acids: êActivity
§ Fasting - êActivity due to unavailability of Acetyl-CoA
§ Hormonal/Covalent modification – Phosphorylation/Dephosphorylation
• Glucagon - Activates Protein Kinase (adds PO4): êActivity
• Insulin – Activates Protein Phosphatase (removes PO4): éActivity
§ Transcriptional control – rate of synthesis of HMGCoA reductase is controlled by sterol regulatory
binding protein (SREBP) when cholesterol levels drop
o Regulation of the LDL Receptor
§ éIntracellular cholesterol: êLDL Receptor synthesis: No more cholesterol uptake
o Regulation of rate of cholesterol esterification and removal of free cholesterol
§ Cholesterol à Bile Acids
§ Cholesterol à Cholesteryl Esters via ACAT
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
Hyperlipidemia
II-A
Accelerated
atherosclerosis, premature Normal Clear B-Lipoprotein Statin drugs
coronary artery disease, é
Niacin
corneal arcus, Cholestyramine
tendinousxanthomas on
achilles tendon, tuberous
xanthoma
II-B Fibrate
*same as II-A é é Clear/Slightly Pre-B Statin drugs
Turbid B-Lipoprotein Niacin
III
Palmarxanthoma on hands
and fingertips, Turbid, creamy Broad B Band Fibrate
tuboeruptivexanthoma over é é
layer sometimes Statin drugs
elbow and knees,
premature atherosclerosis
IV Fibrate
Diabetes, hypertension, Normal/é é Clear/Turbid Pre-B Statin drugs
obesity, atherosclerosis Niacin
V
Abdominal pain, eruptive
xanthoma on extensor Creamy layer on Chylomicrons Niacin
surfaces of arms and legs,
Normal/é é
top, turbid below Pre-B Fibrate
hepatosplenomegaly,
lipidemiaretinalis,
pancreatitis, peripheral
neuropathy
Treatment MOA
• Statins – decreases blood cholesterol levels by competitively inhibiting HMG-CoA Reductase
thus inhibiting cholesterol biosynthesis
• Niacin – decreases VLDL & stimulates reverse cholesterol transport by HDL
• Cholestyramine – sequesters bile acids & excretes them to facilitate bile acid synthesis
from cholesterol to decrease cholesterol levels
• Fibrates – reduces VLDL production & increasing TAG clearance from the blood
• Ezetimibe – inhibits GI cholesterol absorption by ATP-binding cassette transporter inhibition
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
REACTION SUMMARY:
Nitroprusside
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
o Reagents
§ RGT1: BUN Enzyme Reagents
• Phosphate Buffer (pH 7.00) – to maintain optimum pH of Urease
• Sodium Salicylate – condenses with Ammonia to form colored compound
• Sodium Nitroprusside – catalyzes formation of colored compound
• EDTA – chelates metal ions
§ RGT2: Color Developing Reagent
• Phosphate Buffer (pH < 13.00) – maintains constant alkaline condition for color
dev’t
• Sodium Hydroxide – base, donates OH ions for an alkaline environment
• Sodium Hypochlorite – condenses with Ammonia to impart color
§ ENZ: Enzyme Concentrate
• Urease – catalyzes the hydrolysis of Urea to Ammonia & CO2
§ STD: Standard Reagents – for us to be able to have standard readings for sample
computation
• Urea: 80mg/dL (13.3mmol/L)
• BUN Equivalent: 37.28mg/dL (6.2mmol/L)
• Sodium Azide: 0.0095%
o Normal Value
§ Urea = 10 – 50 mg/dL (1.7 - 8.3 mmol/L)
§ BUN = 4.66 – 23 mg/dL (0.79 – 3.86 mmol/L)
o Computation
§ Absorbance of the sample / Absorbance of the Standard (A /A ) SX STD
§ (A /A ) x factor
SX STD
§
We can get both Urea concentration & BUN concentration from the same sample
§
We can also interconvert from Urea concentration à BUN concentration and vice versa
• [BUN] = 0.466 x [Urea]
• [Urea] = 2.14 x [BUN]
• Indications of BUN Measurement
o éBUN (Uremia)
§ Azotemia (Kidney Malfunction): Chronic nephritis, Glomerulonephritis, Polycystic kidney
§ Urinary Tract obstruction – increase back diffusion of Urea to renal tubules
§ Heart Failure
§ Dehydration -
§ High-Protein Diet
§ Muscle wasting – during starvation, body proteins will be broken down & [Urea] will increase
o êBUN
§ Liver Damage/Disease – Urea Cycle impairment: Urea synthesis is impaired: No BUN
§ Malnutrition/Starvation: Low Protein Intake – No protein to catabolize: No BUN
§ 2 /3 Trimester of Pregnancy
nd rd
§ Overhydration
§ Alcoholism
o Variations
§ Adult > Children – growing children use amino acids for synthesis, not breakdown: growth
§ Male Adult > Female Adult – higher protein catabolism in male vs female
• BUN to Creatinine Ratio
o differential diagnosis
§ Creatinine in blood also tells how well the kidneys are working
§ High Creatinine = Kidney Malfunction
o éBUN, Normal Creatinine
§ relative decrease of blood flow to the kidney (as seen in heart failure or dehydration)
without indicating any true injury to the kidney.
o éBUN, éCreatinine
§ Kidney malfunction
• Drugs that interfere with BUN measurement
o False Increase: Allopurinol, Aminoglycosides, Amphotericin B, Carbamazepine, Cephalosporins, Cisplatin, Colistin, High-
dose aspirin, Indomethacin, Methotrexate, Methyldopa, Penicillamine, Probenecid, Propranolol, Rifampin, Diuretics
o False Decrease: Chloramphenicol, Streptomycin
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
• Transamination reactions are important in metabolism because of their function in the synthesis & degradation of
amino acids
• AA -> a-KA -> TCA -> Energy source/Gluconeogenesis
• Compartmentalized intracellularly in specific organs
o Pressence in the Blood means
§ cellular destruction – Hepatocellular Disorders, Skeletal muscle disorders
§ tissue infarction – Myocardial infarction
§ necrosis
• Specimen: Serum, Heparinized Plasma or EDTA Plasma. Avoid Hemolysis
• Transaminases to be tested
University of Santo Tomas Biochemistry
Faculty of Medicine and Surgery John Mark Villena
Class of 2017 Section D
AST/ALT
• Normal Values
• Differential Dx
o AST = marker in both liver and heart
§ Pulmonary Embolism
§ Congestive Heart Disease
§ Hepatocellular disorders
§ Viral Hepatitis
§ Cirrhosis
§ Skeletal muscle disorders
§ Inflammatory conditions
o ALT = marker only in the liver
§ Hepatocellular disorders
§ Acute Inflammatory disorder (liver)