6963
experiments. It should further be noted that, from the standpoint of the charge of the proposed S N ~ C B intermediate, the competition of NOz- with water might be expected to be less favorable for Co(D2H)(N02)-
than for C O ( N H ~ ) ~ ( N H ~ )In ~ +any . case, the mechanistic ambiguity relating to possible contributions from S N ~S, N ~ C Band , SN~CB paths to the base hydrolysis of CO(DH)~(NO~)B~remains to be resolved.
Mechanism for the Proton-Transfer Reactions of a Peptide Hydrogen in Copper ( 11) Triglycine Gordon K. Pagenkopf' and Dale W. Margerumz
Contribution from the Department of Chemistry, Purdue University, Lafayette, Indiana 47907. Received June 26, 1968 Abstract: The rate of interconversion of CuH-zL- and CuH-lL (where L is triglycine and the protons are ionized from the peptide linkages) is much slower than normal acid-base reactions. The rate constant for the reaction of H 3 0+ with CuH+L- is 4.9 X lo6M-l sec-', and the value for the reaction of OH- with CuH-lL is 2.5 x 104 M-1 sec-1. The reason proposed for the slower rates is the necessity to break and rearrange coordinate bonds to copper. The proton addition is general acid catalyzed and proton removal is subject to base catalysis. Two reaction paths are proposed. The copper-imide bond in CuH-?L- is not easily broken and tends to dissociate after the addition of a proton to the peptide nitrogen.
C
opper(I1) complexes of short-chain polypeptides promote the ionization of the peptide hydrogens. 3-g In the present work the speed and mechanism of this type of proton-transfer reaction is examined for copper(I1) triglycine which forms CuH+L (or CuA) and CUH-~L- (or CUB-) with the loss of one and two protons, respectively, from triglycine (L-). The pK, values for these ionizations are 5.4 and 6.6, and from infrared studies in D20 it was concluded that the structure of CUH-~L-can be represented by I.9 The crystal structures of disodium tetraglycinocuprate(I1) and of sodium triglycinocuprate(I1) (NaCuH-L.H20) have
0, I \ (-1
attached to two copper atoms and each copper atom is bonded to two different peptide molecules. The copper is coordinated to the carbonyl oxygen of the peptide link.6 Structures have been suggested for CuL+ and for CUH-~Lin solution where the -NHgroup of the peptide link is c ~ o r d i n a t e d . ~Our kinetic evidence suggests that the proton-transfer reactions between CuH-Land CuH-lL involve coppernitrogen dissociation and that CuH-1L has carbonyl oxygen coordination. The reaction of H30+with CuH-L- to form CUH-~L is much less than the diffusion-controlled rate. l 1 This proton-transfer reaction is general acid catalyzed and can be studied in the presende of ethylenediaminetetraacetate ion (EDTA) which does not react as a nucleophile with CuH-L- but does react with CuH-lL. The reactions of CuH-'L with bases were studied directly using a pH-jump method. Experimental Section Kinetic runs were followed using stopped-flow spectrophotometers. One instrument was described earlierI2 and the other was a
0
Durrum-Gibson stopped-flow, Durrum Instrument Corp., Palo Alto, Calif. Both instruments were thermostated at 25.0 =t0.1 The reaction between copper triglycine and EDTA was followed the peptide nitrogens coordinated to ~ o p p e r . lo ~ , ~ ,by the disappearance of CuH-2L- which has a much higher molar When copper triglycine is crystallized from acid absorptivity than CuH-lL or CuEDTA2- at 555 mp. This wavelength was used for reactions at pH 6.0-7.5 where a significant persolutions to give CuLCl. 1.5Hz0, the peptide chain is centage of the copper triglycine is present as CuH-,L. Above pH (1) Abstracted from the Ph.D. Thesis of G . K. P., Purdue University, 7.5 the reactions were followed at 235 mp. June 1968. Triglycine was obtained (chromatographically homogeneous) (2) Address correspondence to this author. from Mann Research Laboratories (New York, N. Y.)and was used (3) H. Dobbie and W. D. Kermack, Biochem. J . , 59, 246, 257 (1955). without further purification. A 9.85 X M stock solution of (4) A. R. Manyak, C. B. Murphy, and A. E. Martell, Arch. Biochem. Cu(ClO& was prepared from the twice-recrystallized salt and stanBiophys., 59, 373 (1955). dardized against EDTA. The copper(IIfitrig1ycine complex was (5) T. Cooper, H. C. Freeman, G. Robinson, and J. Schoone, Nature, prepared for each series of reactions by mixing copper(I1) and tri194, 1237 (1962). (6) H. C. Freeman and M. R . Taylor, Proc. Chem. Soc., 88 (1964). glycine using a 2Z molar excess of triglycine. The concentration (7) W. L. Koltun, R. H. Roth, and F. R. N. Gurd, J . Biol. Chem., of copper(I1) triglycine used for a kinetic run ranged from 2 x
I (CuH-zL-)
O,
238, 124 (1963). (8) M. K. Kim and A. E. Martell, Biochemisfry, 3, 1169 (1964). (9) M. K. Kim and A. E. Martell, J . A m . Chem. Soc., 88, 914 (1966). (10) H. C. Freeman, J. C. Schoone, and J. G. S h e , Acta C r y s f . , 18, 381 (1965).
Pagenkopf, Margerum
(11) G. K. Pagenkopf and D. W. Margerum, J . A m . Chem. Soc., 90, 501 (1968). (12) D. W. Margerum and J. D. Carr, ibid., 88, 1639 (1966).
Proton Transfer of Peptide Hydrogen in Copper(Il) Triglycine
6964
0
Figure 1. The effect of acidity on the rate of protonation of CUH-~L-. The observed first-order rate constant is corrected for the general acid catalysis of EDTA and buffer. The solid line is calculated from kH = 4.9 X lo6 M-l sec-l and kd = 0.12 sec-’ (see eq 5 ) : 25.0”, p = 0.10. 10-6 to 2 X M depending on the wavelength and the cell path of the stopped-flow device (2 or 20 mm). Ionic strength was maintained at 0.10 M with NaCIOa, and the hydrogen ion concentrations were calculated from pH measurements using -log [H+] = pH -0.11,13 and hydroxide ion concentrations were calculated from pK, = 13.78. Buffers were prepared from boric acid, 2,6-lutidine, and tris(hydroxymethy1)aminomethane (Tris). The buffer concentrations to 1.25 X M. ranged from 4.0 X The acid species of EDTA and of the buffers contribute to the EDTA reaction with CuH-zL-. The rate constants for each species were determined by observing the rate as a function of the concentration of one acid while the concentrations of the other acids remained constant. The observed rates required stopped-flow techniques but were sufficiently slow to permit an excess of the acid species, The rate expression is
Figure 2. Effect of borate ion concentration on the rate constant ko - k o ~ [ o H - ]for the reaction of base with CUH-~L. The slope equals /CH*BO~and the intercept equals kHto.
does agree with H2EDTAZ- acting as a better protontransfer agent. The same behavior was observed for truns-1,2-diaminocyclohexanetetraacetate (CyDTA) which, because of steric requirements, cannot react readily by a nucleophilic mechanism. Other acids also catalyze the reaction and the mechanism is given in eq 2 and 3 where the reaction of the general acid (HX) in eq 2 is the rate-determining step. knx
+ HX CUHIL + Xfast + HEDTA3- + H+ +CuEDTA2- + H L
CUH-ZLCuH-lL
Results Reaction of CuH-2L- with Acid. The rate of reaction of CuH-L- with EDTA in the p H region where monoprotonated EDTA is the predominant species had no dependence upon the EDTA concentration. However, in the pH region where a significant fraction of EDTA was diprotonated, the rate was responsive to the EDTA concentration. This behavior is not in agreement with EDTA acting as a nucleophile because HEDTA3- is more reactive than HzEDTA2-,I4 but it (13) R. G. Bates, “Determination of pH,” John Wiley and Sons, Inc., New York, N. Y . , 1964, p 74.
(3)
The experimentally observed first-order rate constant, ko, is the sum of the contributions of all the species to reaction 2. ko
where k a is a function of the general acid concentrations. For each acid several pH values were used to ensure the correct species assignment. For the reactions of CuH-,L with bases the pH of the copper(I1) triglycine solution was adjusted to about 6.8. At this pH only CUH-~L-and CuH-lL are present in the solution and have a 60: 40 ratio. This solution was mixed by stopped-flow with a solution of the desired proton acceptor. The pH of the latter solution was at least 1.5 units higher than that of copper(I1) triglycine. The rates were followed by observing the appearance of CUH-~L-at 555 rnl.
(2)
=
(4)
C~HX[HX] nx
A summary of the ICHX values is given in Table I. Table I. Rate Constants for Proton Transfer from General Acids to CUH-~L-(25.0”. U. = 0.10 M NaClOA General acid
kHx, M-’ sec-l
Log kHx
pK,
ref
HKyDTAaH~EDTAZH.lutidine+ H Trisf
4.4 X 10’ 3.1 x 103 3.9 X l o a 17 2.2 4.9 x 106 3.2 X lo-‘
3.64 3.49 2.56 1.22 0.35 6.69 -4.5
6.12 6.16 6.75 8.10 9.00 -1.74 15.52
a
HaO+ Hz0
a
b a a
a L. G. Sillen and A. E. Martell, “Stability Constants of Metal-Ion Complexes,” 2nd ed, The Chemical Society, London, 1964. * H. C. Brown and X. R. Mihm, J. Am. Chem. SOC.,77, 1723 (1955).
The dependence of the exchange rate of HaOf is shown in Figure I. These data were obtained by subtracting the HzEDTA2- and the buffer contri(14) D. B. Rorabacher and D. W. Margerum, Inorg. Chem., 3, 382 (1964).
Journal of the American Chemical Society / 90:25 / December 4, 1968
6965 P I,-,
0; 1-1
APK
Figure 3. Proton-transfer rate constants as a function of ApK. The points (0)for HX and CUH-~L-are from left to right HsB03, H . Tris+, H .lutidine+, H2EDTA2-, HzCyDTA2-, and H30+. The points ( 0 )for X- with CUH-~Lare from left to right H2B03- and HzO. The points along the solid curves are for the I s I1 @ 111 a IV mechanism given in Figure 4. The points 0 for HzO CuH-*L- and for OHCUH-~Lare for the I e V s IV mechanism and are not expected to be on the curve.
+
+
(PI
Figure 4. Proposed mechanism for the proton-transfer reactions of copper(I1) triglycine.
Discussion Mechanism of Acid Reaction with CuH-,L-. Log kHX is plotted against ApK (pKacceptor- PKdonor) in butions from the observed rate constant, eq 5. The Figure 3. The limiting value of kHX for the reacsolid line in Figure 1 was calculated from the rate tion with H 3 0 + is four orders of magnitude less than the normal diffusion-controlled rates with hydrogen ion.16 The slope, CY = d(1og kHx)/d(ApK), equals unity even when ApK = 0, whereas in normal protontransfer reactions CY = 0.5 in this region. The fact that a = 1 indicates that the proton must be transferred constants (kH and kd) and the hydrogen ion concentrafrom HX to CUH-~L-before the rate-determining step. tion, The valuel5 of kH is 4.9 X IO6 M-' sec-' and A mechanism is proposed in Figure 4. In this mechthe kd is 0.12 sec-I. The constant kd is attributed to anism the proton is transferred to the peptide nitrogen a molecular rearrangement of CUH-~L-to a form that rather than to the carbonyl oxygen of the peptide can react directly with EDTA, the rate being limited link because the latter transfer would tend to stabilize by the rearrangement. It is possible that H 2 0 and the enol form rather than give CuH-lL. This pathway HEDTA3- could contribute to kd, but the predicted also has been shown to be unproductive in enol to keto contributions from these species are all much less than reactions of acetylacetone16 and diacetylacetone. 17 kd and believed to be insignificant. The peptide nitrogen next to the carboxylate group is Reaction of CUH-~Lwith Base. The rate of reaction assumed to be the point of attack. The carboxylate of CuH+L with H2B03- to form CuH-L- is dependent end of the complex is more labile and nuclear magnetic upon the base concentration as shown in Figure 2. resonance studies with triglycine have shown that The slope is kHzBOa and equals 2.1 f 0.3 X l o 2 M-' the peptide nitrogen nearest to the carboxylate group sec-l. The value of the intercept, 2.5 + 0.2 sec-l, is the is the most basic. rate constant for the reaction of water with CuH-lL In Figure 4 the path I + I1 -+ I11 + IV is proposed or as a second-order rate constant equals 4.5 X le2 for acids with the exception of water where V is believed M- see- l. to be an intermediate as discussed in the next section. The rate constant for the reaction of CuH-1L with The forward rate constant for HX can be expressed hydroxide ion also was determined by a pH-jump by eq 6 and the reverse rate constant, kx, by eq 7. method. The hydroxide ion concentration was varied to 38 X l e 4 M and the k O H value is from 4.3 X kik2k3 2.5 X lo4 M-l sec-l. Rate constants for CuH-lL kHX = (6) k-ik--9 k-ik, kzk3 acting as an acid are listed in Table 11.
+
Table 11. Rate Constants for C U H - ~ LActing as an Acid (25.0", I.( = 0.10 M NaC104) Base, X-
k x , M-l sec-l
HzB03OHHzO
2 . 1 f 0.3 X lo2 2.5 X lo4 4.5 i 0.3 x
(15) In ref 11 we reported the value for k a to be 6.6 X 106 M-1 sec-I.
However, this was based on pH measurements and the more correct value is 4.9 X 106 M - 1 sec-1 based on hydrogen ion concentrations.
+
For both reactions stationary-state conditions are applied to intermediates I1 and 111. These two rate constants, kHX and kx, can be simplified because the equilibrium between I1 and I11 lies to the left. The nitrogen atom in structure I1 is a very weak base and (16) M. Eigen, Angew. Chem., 75, 489 (1963). The present work follows the theoretical treatment given in this reference. (17) J. Stuehr, J . A m . Chem. SOC.,89, 2826 (1967). (18) M. Sheinblatt, ibid., 88, 2123 (1966).
Pagenkopf, Margerum / Proton Transfer of Peptide Hydrogen in Copper(IZ) Triglycine
6966
there are no major changes in configuration in structure I11 so that kl >> k, except for H30+. There are major electronic and structural rearrangements between I11 and IV; thus kl >> k3 and k-l >> k3 and eq 6 and 7 reduce to kHX = K1K,k3 and kx = k+, respectively. This is consistent with the data shown in Figure 3 where a = 1 and p = 0 in the region of ApK = 0 (/3 = d(1og kx)/d(ApK) and a /3 = 1). A p value of zero often implies that the reverse rate is diffusion controlled, but this is not necessary for the CuH-lL reaction with X- because kF1 and k-, are much greater thank+ The rate of formation of I1 should be diffusion I11 controlled so that kl is large. Although step I1 must change the peptide nitrogen to a tetrahedral configuration, this reaction should be fast compared to the rearrangement in step I11 -t IV. Reactions I1 + I11 could be faster than the corresponding reaction of the enol form of acetylacetonate ion because nitrogen is capable of hydrogen-bonding. The reaction of H30+with acetylacetonate has a rate constant of 1.2 X IO7 M-l sec-l at 120.16 The kinetics of hydroxide ion reaction with CuH-lL necessitate a structure for CuH-lL which differs from 111. The structure proposed is shown in IV which agrees with coordination of the carbonyl oxygen found in crystals. Therefore the transition from I11 to IV involves dissociation and rearrangement of copper-ligand bonds. Changing the metal from copper to nickel causes a decrease in the values of kHX despite a more favorable ApK and is consistent with the proposed type of change in coordination. l 9
+
-
0
IJZ (CuH-, L) It has been pointed out that an equilibrium preceding proton transfer can account for rates that are less than diffusion controlled. 1 6 , 2 0 However, a prior dissociation of the -N--group from copper followed by proton transfer can be ruled out. The imide group would be a very strong base, and its reaction with HX would not depend on the acidity of HX. Hence a would approach zero rather than the value of 1 found in this work. The effect of ApK on kHX is largely associated with Kz (= k2/k,) while the values for Kl ( = kl/k-l) should be similar to values for outer-sphere complexes. From the electrostatic attraction with the CUH-~L- the acids H.lutidine+ and H.Tris+ should have larger KI values than HzEDTA2- and H,CyDTA2-. However, the kHX values for H2EDTA2- and H2CyDTA2- fall (19) E. J. Billo and D. W. Margerum, submitted for publication. (20) J . N. Br@nstedand K. Pedersen, Z.Physik. Chem., 108, 185 (1923).
above the line of a = 1 in Figure 3 indicating that if k3 is constant the carboxylate groups of EDTA or CyDTA are weakly bonded to the axial position of copper in CuH-L-. Apical interaction with copper(I1) complexes has been reported. Mechanism of Base Reactions with CuH-lL. When ApK is zero kHXmust equal kx. In addition the sum of the slopes a and p must equal unity so the curve for the base reaction with CuH-lL can be constructed from the value of kHXat ApK = 0 and the H 2 0 rate constant taken from Table 11. The reaction of HzB03- with CuH-lL gives a rate constant of 210 M-l sec-l in agreement with the constructed curve with k = 500 when p = 0. A somewhat unique situation exists in that the reaction between HzB03- and CuH-lL, and the reaction between and CuH-,L- could each be measured independently. This is the case because the latter reaction was driven by the subsequent reaction with EDTA. The p curve in Figure 3 corresponds to I11 4 I1 I path and the rate-determining the IV step is k-3. The ratio of kHx/kx agrees within a factor of 2 with the equilibrium ratio of Ks(HX)/Ka(~u~-IL) for H 3 0 + and for H3B03. The reaction of CuH-1L with hydroxide ion is too fast by a factor of 50 to be accounted for by path 1V -t I11 -t I1 4 I, and the IV V 4 I mechanism in Figure 4 is proposed. Cooper(I1)-catalyzed ester hydrolysis proceeds through an activated intermediate which has the carbonyl oxygen coordinated to the metal.?' A similar intermediate is proposed for the hydroxide ion reaction, Coordination of the carbonyl group would labilize the hydrogen atom bonded to the peptide nitrogen, thus facilitating the removal of the proton before copper is coordinated to the imide nitrogen. Applying the steady-state condition to species V gives
- -
-
The extent to which copper coordination to the carbonyl group labilizes the peptide proton cannot be definitely assigned, but the pK, must be quite large. Hydroxide ion apparently is a strong enough base, with the aid of copper coordination, to extract the proton from the peptide link to form structure V. Other bases will have a much smaller ratio of k-,/k4 and therefore will not react by this path. The value of k-, should approach the diffusioncontrolled limit of 1O1O M-' sec-1 because there is little rearrangement between structures IV and V. Under these circumstances quite weak acids (pK = 11-12) still react at the diffusion limit. It can be seen from eq 8 that in order for k-? >> l o 4 M-I sec-I, k4 must be much larger than k+. Equation 9 follows therefore
from the equilibrium constant of IV and I. The value for kHnO in Table I and in Figure 3 is k5/55.5 M-I sec-l. This point falls above the a slope of unity because it is for the I V -P IV mechanism rather than the I I1 -t I11 + IV mechanism.
-
(21) I
