2 Структура и функция белка (1160071), страница 41
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Solutions of NADH, but not NAD+, absorb light at340 nm. This property is used to determine theconcentration of NADH in solution by measuringspectrophotometrically the amount of light absorbed at 340 nm by the solution. Explain howthese properties of NADH can be used to design aquantitative assay for lactate dehydrogenase.6. Estimation of Vmax and Km by Inspection Although graphical methods are available for accurate determination of the values of Vmax a n d Km ofan enzyme-catalyzed reaction (see Box 8-1), thesevalues can be quickly estimated by inspecting values of Vo at increasing [S].
Estimate the approxi-From these data determine by graphical analysis(see Box 8-1) the values of Km and Vmax for thisenzyme preparation and substrate.9. The Turnover Number of Carbonic AnhydraseCarbonic anhydrase of erythrocytes (Mr 30,000) isamong the most active of known enzymes. It catalyzes the reversible hydration of CO 2 :H 2 O + CO2H 2 CO 3which is important in the transport of CO 2 fromthe tissues to the lungs.(a) If 10 /xg of pure carbonic anhydrase catalyzes the hydration of 0.30 g of CO 2 in 1 min at37 °C under optimal conditions, what is the turnover number (kcat) of carbonic anhydrase (in unitsof min" 1 )?(b) From the answer in (a), calculate the activation energy of the enzyme-catalyzed reaction(in kJ/mol).Chapter 8 Enzymes(c) If carbonic anhydrase provides a rate enhancement of 107, what is the activation energy forthe uncatalyzed reaction?10. Irreversible Inhibition of an Enzyme Manyenzymes are inhibited irreversibly by heavy-metalions such as Hg 2+ , Cu 2+ , or Ag+, which can reactwith essential sulfhydryl groups to form mercaptides:Enz—SH + Ag*Enz—S—Ag +The affinity of Ag+ for sulfhydryl groups is so greatthat Ag+ can be used to titrate —SH groups quantitatively.
To 10 mL of a solution containing 1.0 mg/mL of a pure enzyme was added just enoughAgNO3 to completely inactivate the enzyme. Atotal of 0.342 /zmol of AgNO3 was required. Calculate the minimum molecular weight of the enzyme.Why does the value obtained in this way give onlythe minimum molecular weight?11.
Protection of an Enzyme against Denaturationby Heat When enzyme solutions are heated, thereis a progressive loss of catalytic activity with time.This loss is the result of the unfolding of the nativeenzyme molecule to a randomly coiled conformation, because of its increased thermal energy. Asolution of the enzyme hexokinase incubated at45 °C lost 50% of its activity in 12 min, but whenhexokinase was incubated at 45 °C in the presenceof a very large concentration of one of its substrates, it lost only 3% of its activity.
Explain whythermal denaturation of hexokinase was retardedin the presence of one of its substrates.12. Clinical Application of Differential EnzymeInhibition Human blood serum contains a class ofenzymes known as acid phosphatases, which hydrolyze biological phosphate esters under slightlyacidic conditions (pH 5.0):oR—O—P—CT + H2OO239retic (increases the production of urine) and totreat glaucoma (reduces excessively high pressurewithin the eyeball).
Carbonic anhydrase plays animportant role in these and other secretory processes, because it participates in regulating the pHand bicarbonate content of a number of body fluids.The experimental curve of reaction velocity (givenhere as percentage of Vmax) versus [S] for the carbonic anhydrase reaction is illustrated below(upper curve). When the experiment is repeated inthe presence of acetazolamide, the lower curve isobtained. From an inspection of the curves andyour knowledge of the kinetic properties of competitive and noncompetitive enzyme inhibitors, determine the nature of the inhibition by acetazolamide.Explain.100r10014.
pH Optimum of Lysozyme The enzymatic activity of lysozyme is optimal at pH 5.2.1000"R—OH + HO—P—0"I50OAcid phosphatases are produced by erythrocytes,the liver, kidney, spleen, and prostate gland. Theenzyme from the prostate gland is clinically important because an increased activity in the blood isfrequently an indication of cancer of the prostategland. The phosphatase from the prostate gland isstrongly inhibited by the tartrate ion, but acidphosphatases from other tissues are not. How canthis information be used to develop a specific procedure for measuring the activity of the acid phosphatase of the prostate gland in human bloodserum?13.
Inhibition of Carbonic Anhydrase by Acetazolamide Carbonic anhydrase is strongly inhibitedby the drug acetazolamide, which is used as a diu-10pHThe active site of lysozyme contains two amino acidresidues essential for catalysis: Glu35 and Asp52.The pKa values of the carboxyl side chains of thesetwo residues are 5.9 and 4.5, respectively. What isthe ionization state (protonated or deprotonated) ofeach residue at the pH optimum of lysozyme? Howcan the ionization states of these two amino acidresidues explain the pH-activity profile of lysozyme shown above?.
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