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Turn Allows This Particular Enzyme to Degrade a Polynucleotide in a Continuous Manner

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Turn Allows This Particular Enzyme to Degrade a Polynucleotide in a Continuous Manner A NEW KINETIC MODEL FOR POLYNUCLEOTIDE METABOLISM BY HARRISON M. LAZARUS,* MICHAEL B. SPORN,t AND DAN F. BRADLEY: NATIONAL CANCER INSTITUTE, AND NATIONAL INSTITUTE OF MENTAL HEALTH, BETHESDA, MARYLAND Communicated by C. B. Anfinsen, May 22, 1968 Several exonucleases, such as the exoribonuclease from Ehrlich ascites tumor cells, I Escherichia coli ribonuclease 11,2 and bacterial polynucleotide phosphoryl- ase,3, 4 have recently been shown to remain complexed with an individual poly- nucleotide molecule while the enzyme continuously and almost completely de- grades the polymer to mononucleotides. The behavior of these exonucleases is in marked contrast to that of the exonuclease from snake venom2' 3 and exo- nuclease I from E. coli.5 When snake venom exonuclease hydrolyzes polynucleo- tides, the enzyme-substrate complex dissociates into separate entities between successive hydrolytic steps, and the next phosphodiester bond hydrolyzed comes from a polynucleotide molecule taken at random from the molecules in the vicin- ity of the enzyme. However, when Ehrlich ascites tumor cell exoribonuclease, E. coli ribonuclease II, and bacterial polynucleotide phosphorylase degrade polynucleotides, the polynucleotide apparently does not dissociate from the enzyme after cleavage of the terminal mononucleotide, but rather the enzyme and shortened polynucleotide move relative to each other so as to bring the next phosphodiester bond into position for cleavage. By means of such procession, an entire polynucleotide molecule may be degraded without ever leaving the sur- face of the enzyme. The presumption of a processive step implies that after a hydrolytic step, the enzyme and shortened polynucleotide are still held to- gether by one or more bonds. We wish to point out that the processes of dissocia- tion and procession of the polynucleotide should not be considered as mutually exclusive; after the first cleavage step, there will in general be a probability for dissociation and a probability for procession, the values for which are related to the rate constants for the two processes. We would like to present a formal kinetic model of exonucleolytic action that includes both classes of exonucleases as special cases for limiting values of the k6inetic constants. This model has been used to design kinetic experiments witl the exoribonuclease from Ehrlich ascites tumor cells. The data yield information about: (1) the number of bonds between the enzyme and the polynucleotide being degraded; (2) the energetic contribution of each bond; and (3) the free- energy drop causing procession of the polynucleotide on the enzyme, which in turn allows this particular enzyme to degrade a polynucleotide in a continuous manner. Although the kinetic model is applicable to both random and continuous exo- niucleolytic degradation, it will be presented and illustrated (Fig. 1) with empha- sis on the special case of continuous degradation: The enzyme is assumed to bind a substrate, such as polyadenylic acid (Poly A), of chain length n (step 1, Fig. 1). The binding is assumed to be between m filled binding places on the enzyme and m places on the polymer. The chain length, n, of the polymer is greater than the number of binding places, m. The hydrolysis of the terminal 1503 Downloaded by guest on September 27, 2021 1504 BIOCHEMISTRY: LAZARUS ET AL. PROC. N. A. S. Enzyme FIG. l.-Schematic representation k and kinetic model of an exonuclease 1 E+Ani EmAn Pydeyc i degrading a polymer. E represents k-i an exonuclease, A. is a polynu- Polyodenylic Acid cleotide (such as Poly A) of chain length n, EmAn is the enzyme- Enzyme polynucleotide complex in a con- k2 figuration capable of liberating 2 EmAn )EmiAn+Ai the terminal nucleotide, with m 'lip-??....?+AMP as the number of enzyme-poly- Polyodenylic Acid nucleotide bonds. Em-iAn-i is Enzyme the enzyme-polynucleotide com- A; plex, with the terminal nucleotide k3 3 l-mAn- liberated as A1. Em.-An-i repre- Em-imn k-3 sents a complex no longer capable Polyodenylic Acid of releasing another mononucleo- tide until it returns to the confor- Enzyme mation of EAn,1 == E,3A.. Ir k4 is an oligonucleotide of chain 4 E+Ir'EmIr length r, which is not hydrolyzed. k-4 0....ie Emar represents the enzyme-oligo- 01ligonucleotide nucleotide complex. mononucleotide is not kinetically reversible (step 2, Fig. 1). The polynucleo- tide can now move over one unit to fill up all the enzyme binding points (step 3, Fig. 1). An oligonucleotide of chain length r that is not degraded by the exo- nuclease can be bound reversibly to the enzymatic site and act as an inhibitor (step 4, Fig. 1). Thus, the over-all reaction for substrates is ki k2 E E,-1A + Al. + An EmAnk3l n4- k-a In setting down the kinetic model, we have assumed that the hydrolytic step is kinetically irreversible and that the polymers are sufficiently long so that EmAni- has the same kinetic behavior as EmAn; i.e., EmAn = EmAni. The differential rate equations for this model are as follows: d [A1 I= dt k2[EmAn] =V, (1) d[EmAn] = ] dt ki[E][An] -k-i[EmAn] -k2[EmAn] -k-3[EmAn-l +k3[EmlAnj] (2) d[Em-iAn-1] = k2 dt [EmAn] +k-3[EmAnI] -k3[EmiAn..], (3) d[EmIr] = dt k4[E][Ir] -k.4[EmIrI]. (4) Mlaking the steady-state assumption that the concentration of all intermediate species (EmAn, EmiAn-i, EmIr, with EmAn EmAn-) are time-independent from (3), [Em-iAn-i] = [(k2 + k4)/k3] [EmAn]; from (2) and (3), [E] = (k-1 [EmAnI)/ Downloaded by guest on September 27, 2021 VOL. 60, 1968 BIOCHEMISTRY: LAZARUS ET AL. 1505 (k1 [An]); and from (4) and above, [EmIr] = (k-1 k4 [EmAn] [Ir])/(k1 k4 [An]) Substituting into the conservation equation [Eo] = [E] + [EmIr] + [EmAn] + [Em-iAnj], [Eo] = [EmAnI (1 + k4A I + k2+k3 + k1k4A ]) From (1), 1 1 1 (1 + k-1 + k2+k-3 + k-1k4[Ir] V k2[EmAn] k2[Eo] \ k[An] k3 kik4[An]J/' therefore, 1( + k2 + ()+ V (+ KI 1A where Vmax = k2 [Eo], Keq = k-l/ki, Ki = k4/k4. The model does not require the enzyme to degrade an individual polynucleo- tide molecule continuously, but allows it to do so. However, after step (3), the EmAn complex can dissociate to Em and An by reversing step (1). In contrast, if step (2) were replaced by EmAn -- E + An-1 + A1, then the enzymatic degrada- tion would have to be random. However, in the present model, whether or not the enzyme is continuously degrading the same polymer molecule depends only on the ratio of k2/k-1. A plot of 1/v versus 1/An with Ir = 0 gives a Lineweaver-Burk6 plot (Fig. 2A). In plotting 1/v versus Ir, as done by Dixon7 (Fig. 2B), at two different concentra- tions of An the same 1/v will be obtained at the same Ir concentration when Ir = -Ki. Ki can also be determined, for Ki = -Ir at 1/v = 1/Vmax [1 + A B Keq A Slope- I/v Vmax I/v I/Vmx (I+ I/Vmax(I+ k 3 0 ° I/An Ir -Kj FIG. 2.-Graphic representation of kinetic constants. (A) A Lineweaver-Burk plot. An is the concentration of a polymer of chain length n, v is the velocity. The derivation of the slope and y intercept are given in the text. (B) A Dixon plot. Ir is the concentration of inhibitor of chain length r, and the derivation of Ki is in the text. An' and An2 represent two different concentrations of polyitucleotide of chain length n. Downloaded by guest on September 27, 2021 1506 BIOCHEMISTRY: LAZARUS ET AL. PROc. N. A. S. (k2 + k-3)/k3] (Fig. 2B). The Ks's of a set of I,'s can be used to find the maxi- mum number of monomer units (r) necessary to saturate the principal binding of an exonuclease; i.e., an r is sought such that r + q, q _ 1, will give no greater inhibition of the enzyme than r as reflected in the Ki. In the present work, the model is used to evaluate the number of principal binding points, as well as the energetic contribution of each bond, for the exoribo- nuclease from Ehrlich ascites tumor cells.' This enzyme attacks polynucleo- tides, such as Poly A, from the 3'-OH end, liberating 5'-mononucleotides. Oligo- nucleotides terminated by a 2', 3'-cyclic phosphate, such as (Ap)r-,A-cyclic-p, are not degraded by this exoribonuclease and are competitive inhibitors, as well as analogues, of the substrate Poly A. The K/'s for a set of these oligonucleo- tides, I, (r, chain length, from 1 to 8), were determined with Poly A as substrate for exoribonuclease. Experimental Procedure.-Materials: Poly A-H3 and oligonucleotides were obtained from Miles Laboratories, Elkhart, Indiana. The exoribonuclease was prepared from Ehrlich ascites tumor nuclei.' Analytical methods: (a) Enzymatic assays: The standard assay mixture (0.5 ml) was as follows: 0.1 M Tris-HCl, pH 7.7; 5mM MgCl2; 400,ug/ml BSA; 0.4mM dithio- threitol; 0.84mM or 1.68mM or2a52mM Poly A-H3 (mononucleotide equivalent), spec. act. 0.25 Ac/,uM; 0.0-0.048 M (Ap),_-A-cyclic-p, varying with the chain length; exo- ribonuclease 0.1-0.3 units/ml. A unit of enzyme is defined as that amount which forms 1 gmole of AMP per hr with 3mM Poly A as substrate.' After 30 min at 370, the reac- tion was stopped by the addition of 0.5 ml ice-cold 0.8 M perchloric acid. The tubes were centrifuged for 30 min at 1900 X g, and the radioactivity of 0.4 ml of the clear supernatant was measured.
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