2 Структура и функция белка (1160071), страница 40
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To be active, some enzymes require a chemicalcofactor, which can be loosely or tightly bound.Each enzyme is classified according to the specificreaction it catalyzes.Enzyme-catalyzed reactions are characterizedby the formation of a complex between substrateand enzyme (an ES complex). The binding occursin a pocket on the enzyme called the active site.The function of enzymes and other catalysts is tolower the activation energy for the reaction andthereby enhance the reaction rate.
The equilibriumof a reaction is unaffected by the enzyme.Part II Structure and CatalysisThe energy used for enzymatic rate enhancements is derived from weak interactions (hydrogenbonds and van der Waals, hydrophobic, and ionicinteractions) between the substrate and enzyme.The enzyme active site is structured so that manyof these weak interactions occur only in the reaction transition state, thus stabilizing the transitionstate. The energy available from the numerousweak interactions between enzyme and substrate(the binding energy) is substantial and can generally account for observed rate enhancements. Theneed for multiple interactions is one reason for thelarge size of enzymes.
Binding energy can be usedto lower substrate entropy, to strain the substrate,or to cause a conformational change in the enzyme(induced fit). This same binding energy accountsfor the exquisite specificity exhibited by enzymesfor their substrates. Other catalytic mechanismsinclude general acid-base catalysis and covalentcatalysis. Details of the reaction mechanisms havebeen worked out for many enzymes.Kinetics is an important method for the study ofenzyme mechanisms.
Most enzymes have somecommon kinetic properties. As the concentration ofthe substrate is increased, the catalytic activity ofa fixed concentration of an enzyme will increase ina hyperbolic fashion to approach a characteristicmaximum rate Vmax> at which essentially all theenzyme is in the form of the ES complex. The substrate concentration giving one-half Vmax is theMichaelis-Menten constant ifm, which is characteristic for each enzyme acting on a given substrate. The Michaelis-Menten equationuKm + [S]relates the initial velocity of an enzymatic reactionto the substrate concentration and Vmax throughthe constant Km.
Both Km and Vmax can be measured; they have different meanings for differentenzymes. The limiting rate of an enzyme-catalyzedreaction at saturation is described by the constantkcaU also called the turnover number. The ratiokcat/Km provides a good measure of catalytic efficiency. The Michaelis-Menten equation is alsoapplicable to bisubstrate reactions, which occur byeither ternary complex or double-displacement(ping-pong) pathways. Each enzyme has an optimum pH, as well as a characteristic specificity forthe substrates on which it acts.Enzymes can be inactivated by irreversiblemodification of a functional group essential for catalytic activity.
They can also be reversibly inhibited, competitively or noncompetitively. Competitive inhibitors compete reversibly with thesubstrate for binding to the active site, but they arenot transformed by the enzyme. Noncompetitiveinhibitors bind to some other site on the free enzyme or to the ES complex.Some enzymes regulate the rate of metabolicpathways in cells. In feedback inhibition, the endproduct of a pathway inhibits the first enzyme ofthat pathway. The activity of some regulatory enzymes, called allosteric enzymes, is adjusted byreversible, noncovalent binding of a specific modulator to a regulatory or allosteric site. Such modulators may be inhibitory or stimulatory and may beeither the substrate itself or some other metabolite.
The kinetic behavior of allosteric enzymes reflects cooperative interactions among the enzymesubunits. Other regulatory enzymes are modulated by covalent modification of a specific functional group necessary for activity, or by proteolytic cleavage of a zymogen.GeneralEvolution of Catalytic Function.
(1987) ColdSpring Harb. Symp. Quant. Biol. 52.A collection of excellent papers on many topics discussed in this chapter.Fersht, A. (1985) Enzyme Structure and Mechanism, W.H. Freeman and Company, New York.A clearly written, concise introduction. More advanced.Friedmann, H. (ed) (1981) Benchmark Papers inBiochemistry, Vol. 1: Enzymes, Hutchinson RossPublishing Company, Stroudsburg, PA.A collection of classic papers in enzyme chemistry,with historical commentaries by the editor.
Extremely interesting.Jencks, W.P. (1987) Catalysis in Chemistry andEnzymology, Dover Publications, Inc., New York.A new printing of an outstanding book on the subject. More advanced.Chapter 8 Enzymes237Principles of CatalysisEnzyme ExamplesHansen, D.E. and Raines, R.T. (1990) Bindingenergy and enzymatic catalysis. J.
Chem. Educ.67, 483-489.A good place for the beginning student to acquire abetter understanding of principles.Anderson, CM., Zucker, F.H., & Steitz, T.A.(1979) Space-filling models of kinase clefts and conformation changes. Science 204, 375-380.Structure of hexokinase and other enzymes utilizing ATP.Jencks, W.P. (1975) Binding energy, specificity,and enzymic catalysis: the Circe effect. Adv.
Enzymol. 43, 219-410.Fersht, A.R. (1987) Dissection of the structure andactivity of the tyrosyl-tRNA synthetase by sitedirected mutagenesis. Biochemistry 26, 80318037.Kraut, J. (1988) How do enzymes work? Science242, 533-540.Lerner, R.A., Benkovic, S.J., & Schulz, P.G.(1991) At the crossroads of chemistry and immunology: catalytic antibodies.
Science 252, 659-667.Schultz, P.G. (1988) The interplay between chemistry and biology in the design of enzymatic catalysts. Science 240, 426-433.KineticsCleland, W.W. (1977) Determining the chemicalmechanisms of enzyme-catalyzed reactions by kinetic studies. Adv. Enzymol. 45, 273-387.Raines, R. T. & Hansen, D.E. (1988) An intuitiveapproach to steady-state kinetics. J. Chem.
Educ.65, 757-759.Warshel, A., Naray-Szabo, G., Sussman, F., &Hwang, J.-K. (1989) How do serine proteasesreally work? Biochemistry 28, 3629-3637.Regulatory EnzymesDische, Z. (1976) The discovery of feedback inhibition. Trends Biochem. Sci. 1, 269-270.Koshland, D.E., Jr. & Neet, K.E. (1968) The catalytic and regulatory properties of enzymes. Annu.Rev. Biochem. 37, 359-410.Monod, J., Changeux, J.-P.
& Jacob, F. (1963)Allosteric proteins and cellular control systems.J. Mol. Biol. 6, 306-329.A classic paper introducing the concept of allostericregulation.Segel, I.H. (1975) Enzyme Kinetics: Behavior andAnalysis of Rapid Equilibrium and Steady StateEnzyme Systems, John Wiley & Sons, Inc., NewYork.A more advanced treatment.Problems1. Keeping the Sweet Taste of Corn The sweettaste of freshly picked corn is due to the high levelof sugar in the kernels. Store-bought corn (severaldays after picking) is not as sweet, because about50% of the free sugar of corn is converted intostarch within one day of picking. To preserve thesweetness of fresh corn, the husked ears are immersed in boiling water for a few minutes("blanched") and then cooled in cold water.
Cornprocessed in this way and stored in a freezer maintains its sweetness. What is the biochemical basisfor this procedure?2. Intracellular Concentration of EnzymesTo ap-proximate the actual concentration of enzymes in abacterial cell, assume that the cell contains 1,000different enzymes in solution in the cytosol, thateach protein has a molecular weight of 100,000,and that all 1,000 enzymes are present in equalconcentrations.
Assume that the bacterial cell is acylinder (diameter 1 /mi, height 2.0 /mi). If the cytosol (specific gravity 1.20) is 20% soluble proteinby weight, and if the soluble protein consists entirely of different enzymes, calculate the averagemolar concentration of each enzyme in this hypothetical cell.238Part II Structure and Catalysis3. Rate Enhancement by Urease The enzyme urease enhances the rate of urea hydrolysis at pH 8.0and 20 °C by a factor of 1014. If a given quantity ofurease can completely hydrolyze a given quantityof urea in 5 min at 20 °C and pH 8.0, how long willit take for this amount of urea to be hydrolyzedunder the same conditions in the absence of urease? Assume that both reactions take place in sterile systems so that bacteria cannot attack the urea.4.
Requirements of Active Sites in Enzymes Theactive site of an enzyme usually consists of apocket on the enzyme surface lined with the aminoacid side chains necessary to bind the substrateand catalyze its chemical transformation. Carboxypeptidase, which sequentially removes the carboxyl-terminal amino acid residues from its peptide substrates, consists of a single chain of 307amino acids. The two essential catalytic groups inthe active site are furnished by Arg145 and Glu270.(a) If the carboxypeptidase chain were a perfecta helix, how far apart (in nanometers) wouldArg145 and Glu270 be? (Hint: See Fig. 7-6.)(b) Explain how it is that these two amino acids,so distantly separated in the sequence, can catalyze a reaction occurring in the space of a fewtenths of a nanometer.(c) If only these two catalytic groups are involved in the mechanism of hydrolysis, why is itnecessary for the enzyme to contain such a largenumber of amino acid residues?5.
Quantitative Assay for Lactate DehydrogenaseThe muscle enzyme lactate dehydrogenase catalyzes the reactionOCH3-C-COO + NADHPyruvatemate value of V m a x and Km for the enzyme-catalyzed reaction for which the following data wereobtained:rsi (M)V0(MM/min)2.5 x IO- 64.0 x io- 61 x lO- 52 x lO- 54 x lO- 5l x io- 42 x io- 31 x io- 2284070951121281391407.
Relation between Reaction Velocity and Substrate Concentration: Michaelis-MentenEquation(a) At what substrate concentration will an enzyme having a kcat of 30 s" 1 and a Km of 0.005 Mshow one-quarter of its maximum rate?(b) Determine the fraction of Vmax that would befound in each case when [S] = iKm, 2ifm> and 10ifm.8. Graphical Analysis ofVmax and Km Values Thefollowing experimental data were collected duringa study of the catalytic activity of an intestinalpeptidase capable of hydrolyzing the dipeptideglycylglycine:2 glycineGlycylglycine + H 2 O[S] (mM)Product formed (/Ltmol/min)1.52.03.04.08.016.00.210.240.280.330.400.45OHCH3—C—COO- + NAD+HLactateNADH and NAD+ are the reduced and oxidizedforms, respectively, of the coenzyme NAD.
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