31P n.m.r. studies of complexes of NADPH and NADP+ with Escherichia coli dihydrofolate reductase

n.m.r, studies of complexes of N A D P H and N A D P ÷ withEscherichia coli dihydrofolate reductase P. J. Cayley, J. Feeney and B. J. Kimber Division ~f Molecular Pharmacology, Natiomd Institute.Ira" Medical Research, Mill Hill, London N W7 1AA, UK

(Received 18 October 1979) We have measured the 31p n.m.r, spectra of N A D P + and N A D P H in their binary complexes with Escherichia coli dihydro[blate reduetase and in ternary complexes with the enzyme and.[blate or methotrexate. The 3~p chemical sh(fi Of the 2' phosphate group is the same in all complexes; its value indicates that it is binding in the dianionic state and its pH independence suggests that it is interaeting sti'ongly with cationic residue(s) on the enzyme. Similar behaviour has been noted previously.Jor the eomplexes with the Lactobacillus casei e n z y m e although the 3~ p sh([? is somewhat d![]erent in this complex, possibly due to an interaetion between the 2' phosphate group and His 64 whieh is not conserved in the E. coli enzyme. For the coenzyme complexes with both enzymes s i p 0 C z 1H z, spin spin interactions were detected (7.5 7.8 Hz) on the 2' phosphate resonances, indicating a P 0 C 2- H 2, dihedral angle of 30 or 330 : this is in good agreement with the value of330 measured in erystallographie studies ~ (Matthews et al., 1978) on the L. casei enzynw. N A D P H M T X eomplex. The pyrophosphate resonances are sh(fied to d([]brent extents in the, various complexes and there is evidence that there is more O - P - O bond angle distortion in the E. coil enzyme complexes than in those with the L. casei enzyme. The q[]ects O]3 ~P 0 C s l H s, spin coupling were detected on one pyrophosphate resonance and indicate that the P 0 Cs Hs, torsion angle has changed by at least ~ 30 on binding to the E. coli enzyme: this is considerably less than the distortion ( ~ 50:) observed previously in the L. casei enzyme complex.

Introduction

al. 9 Bound folate and other impurities were removed from

Dihydrofolate reductase (EC 1.5.1.3) catalyses the reduction of dihydrofolate to tetrahydrofolate using the coenzyme NADPH 2'3. As part of a wider study of ligand binding to dihydrofolate reductase we have used high resolution 1H, 13C and 31p n.m.r, spectroscopy to provide detailed information about the conformation and ionization states of N A D P H and N A D P + bound to the Lactobacillus casei enzyme'*- 7. We have now extended our studies to the complexes of these coenzymes with dihydrofolate reductase from Escherichia colk N A D P H binds one hundred-fold less tightly to this than to the L. easel enzyme and it is of interest to see if n,m.r, studies on the complexes can indicate the origin of this binding difference. In this paper we report 31 p n.m.r, measurements on the coenzyme complexes with E. coil dihydrofolate reductase and compare them with our earlier measurements on the L. casei enzyme 5'7. Because there are only three phosphorus nuclei in the coenzymes, their complexes give rise to 31p spectra which are fairly simple, particularly when compared with t H spectra where there is extensive overlap of the signals from protein and coenzyme protons. E. coli dihydrofolate reductase exists as two isoenzymes which differ in their binding to trimethoprim and methotrexate s but which have similar equilibrium binding constants for N A D P H .

the enzyme using a column of DEAE-23 cellulose equilibrated in 25 mM Tris, 500 mM KCI at pH 8.5 10, The enzyme sample was a mixture of the two isoenzymes present in approximately equal amounts. N A D P H , N A D P + and folate were obtained from Sigma Chemicals and methotrexate was from Nutritional Biochemicals Corp. The 2H20 (99.85 atom% 2H) was from Norsk Hydroelectrisk, 2HCI (99 ato - m .... j,i, ZH) was from CIBA (ARL) Ltd and KOZH was prepared from K O H by exchanging twice with 2H20. All the other chemicals were of Analar reagent grade. The dihydrofolate reductase was prepared for n.m.r. study by lyophilizing twice from 2H20 solution to remove all exchangeable protons and then dissolving in 15 mM bis Tris, 500 mM KC1 and 1 mM EDTA to give a final enzyme concentration of 1.0 to 1.2 raM. For the n.m.r, experiments, 1.5 ml volumes of solution were contained in 12 mm diameter tubes fitted with vortex suppressors. The pH adjustments were made using 0.2 M KO2H and 0.2 M 2HCI and the pH measurements were uncorrected meter readings taken with a Radiometer R26 pH meter equipped with a glass micro electrode. The N A D P H and N A D P + were added as microlitre volumes of concentrated solutions while folate and methotrexate were added as weighed amounts of the solids to the enzyme solutions.

Experimental

N.m.r. spectroscopy

M a t e r i a l s and sample preparation

Dihydrofolate reductase was purified from E. coli B RT500 using a modification of the method of Dann et 0141 8130 80.040251 05502.00 © 1980 I P C Business Press

The alp n.m.r, measurements were made at 40.5 MHz on a Varian XL-100 spectrometer interfaced to a 620i computer operating in the Fourier transform mode. To minimize problems related to pulse breakthrough, the

Int. J. Biol. Macromol., 1980, Vol 2, August

251

31p n.m.r, studies of complexes o [ ' N A D P H amt N A D P * : P. d. Cayley et al. shifts were measured with respect to a 50 mM K 2 H P O 4 solution (pH 8.0) used as an external standard.

a

Results Complexes with N A D P H In the proton noise-decoupled 31p spectrum of free N A D P H (shown in Figure la) the 2'-phosphate signal occurs at low field ( + 0.47 p.p.m, at pH 6.9) while the two p y r o p h o s p h a t e phosphorus nuclei accidentally have the same chemical shift and thus give rise to a single absorption at - 13.78 p.p.m. When the N A D P H is bound to E. coli dihydrofolate reductase, the T - p h o s p h a t e signal shifts 0.70 p.p.m, to lower field and the two p y r o p h o s p h a t e signals shift upfield to different extents (1.82 and 3.73 p.p.m.), as illustrated in Figure lb. Because the two phosphorus nuclei are no longer shielded equivalently, their signals appear as two AB doublets resulting from spin-spin interaction between the phosphorus nuclei (Jp o p = 2 2 4 - 1 Hz). Figure lb clearly shows that the spectrum of the complex of N A D P H with the mixture of E. coli isoenzymes has only one set of three 31p signals, indicating that N A D P H has an identical spectrum when bound to either form of the enzyme; this was subsequently found to be true for all complexes of the enzyme with N A D P H and N A D P + in the presence and absence of substrate analogues. When the pH is varied over the range 5-7 no change is detected in the 31p chemical shifts of the complex; as indicated in Figure 2 this contrasts markedly with the behaviour of the T - p h o s p h a t e 31p chemical shift of free

lj

cp d

I 4,0

I

I

I

I

0.0

-14.0

1

I

I

-18.0

p.p.m.

Figure l (a) 3]p Proton noise decoupled spectrum of 10 mM NADPH at pH 6.9. (b) 31p Proton noise decoupled spectrum of 1 mM NADPH in the presence of 1 mM E. coli dihydrofolate reductase at pH 6.9. (c) 31p Single resonance spectrum of 4 mM NADPH in the presence of 1 mM E. coli dihydrofolate reductase at pH 6.9 and 40C. The signals at +0.47 and - 13.78 p.p.m, are from free NADPH while that at - 1 . 6 p.p.m, arises from a phosphate group in an unknown decomposition product which forms on standing. (d) 31 p Single resonance spectrum of 1.1 mM NADPH in the presence of 1.2 mM L. casei dihydrofolate reductase at pH 6.9 and 4 0 C

-2

-3 5

block-averaging technique was used: typically, 200 transients (acquisition time 0.5 to 1.0 s) were collected in each block and up to 1000 blocks were accumulated. A spectral width of 2 K H z was collected in 2 to 4K data points and the free induction decay was usually multiplied by an exponentially decreasing weighting function to improve the signal-to-noise ratio. The 3xp spectra were obtained with and without proton noise decoupling. The chemical

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Int. J. Biol. Macromol., 1980, Vol 2, August

6

7

8

pH

Figure 2 Variation of chemical shift of 2' phosphate 31p lesonance of NADP + (e, ©) and NADPH ( v v) with pH. The solid symbols refer to the free coenzymes and the open symbols to coenzyme bound to E. coil dihydrofolate reductase (v, NADPH enzyme; ©, NADP + enzyme-methotrexaie). The pH values are uncorrected meter readings "(glass electrode) from D2O solutions

3 1 p n.m.r,

studies of complexes of N A D P H aml N A D P + : P. J. Cayley et al.

Table I 3~p chemical shifts and coupling constants for NADPH and NADP + in their complexes with dihydrofolate reductase at pH 6.9 at 10 C 2' Phosphate Chemical shift (p.p.m.) N A D P H (pH 6.9)" N A D P + ( p H 6.9)" E. coli D H F R NADPH enzyme NADPH methotrexate enzyme NADP + enzyme NADP + methotrexate enzyme NADP + folate~enzyme L. casei D H F R NADPH~nzyme NADPH- methotrexate enzyme NADP + -enzyme"

Pyrophosphate

31p O C-1H2 , Coupling constant (Hz)

Chemical shift (p.p.m.) A B

31p_o_31P

Coupling constant (Hz)

-13.78 (-14.15

-13.78 -14.47)

-15.60

-17.51

21.9±1.0

1.16 1.17

- 15.39 -

-17.01 ( - 15.9)b

22.5±1.0

1.18 1.17

- 16.32 -15.90

- 16.70 -16.71

-13.94

-16.47

- 13.93 - 14.32

- 16.33 - 16.23

0.47 -0.22 1.17

2.66

7.8±1.0

7.5±1.0

2.66 2.72

20.6_+ 1.0

All chemical shifts are quoted in p.p.m, from the standard 50 mM K 2 H P O 4 solution (pH 8.0) at 10 C. Positive shifts are to low field. Chemical shifts have errors +0.05 p.p.m. Values taken from Feeney et al. 5 h Error _+0.5 p.p.m.

N A D P H (pK = 6.1) but is similar to that noted previously for the complex with L. casei dihydrofolate reductase 5'6. Addition of another equivalent of N A D P H to the solution results in extra signals appearing at the frequency positions of those for free N A D P H . This confirms that the N A D P H is binding with a stoichiometry of 1 mol per mol of enzyme and that there is slow exchange on the n.m.r. timescale between free and bound forms of the coenzyme. When the proton irradiation is removed the effects of 3 1 P 1 H spin-spin interactions are clearly visible in the 3 ~p spectra (see Fi,qure I c). The 2'-phosphate signal is now a well-resolved doublet from coupling to the H z, proton (J-p o c 'H2 = 7 . 8 + 1 Hz) and the lower field pyrophosphate resonance shows evidence of coupling to its neighbouring 5' methylene protons (J3,p o c ~H~.q"J3'P O C ~Hs~ >~8 _+2 Hz) while the other resonance shows no detectable sign of proton coupling. The observed splittings could be somewhat less than the actual coupling constants if rapid relaxation of the interacting protons results in a partial decoupling of the spin spin interactions. Fiqure ld shows the single r e s o n a n c e 3 1 p spectrum of the L. casei e n z y m e - N A D P H complex which has been discussed previously 5. By examining the sample at a higher temperature (40"C) than previously used a better resolved spectrum was obtained in which the doublet splitting (7.5 Hz) on the 2'-phosphate signal was clearly resolved. In the ternary complex of E. coil dihydrofolate reductase with N A D P H and methotrexate, the 2'-phosphate 31p signal is in the same position as in the binary complex but appreciable downfield shifts are noted for both pyrophosphate signals (0.2 and 0.5 p.p.m.). All the chemical shift and coupling constant results are summarized in Table 1. The observed coupling constants have similar values in the binary and ternary complexes (see Table 1).

Complexes with N A D P + When one equivalent of N A D P + is added to a 1 mM solution of E. coli dihydrofolate reductase, the 31 p signals are again shifted but, unlike those in the N A D P H complex, they are considerably broadened. The 2'-phosphate signal of bound N A D P + is again shifted downfield by 0.7 p.p.m. However, the N A D P + pyrophosphate signals are too broad to allow them to be characterized properly in the binary complex. Addition of more N A D P + gives rise to additional broad signals at the frequencies corresponding to free N A D P + signals, confirming that there is slow exchange between the bound and free species. The broadening increases with temperature showing that there is an exchange contribution to the linewidths. At 10°C this exchange contribution is ~ 20 Hz, indicating that the dissociation rate constant is ~ 60 s - 1. Addition of one equivalent of methotrexate or folate caused the lines to sharpen (as a result of the increased lifetime of the coenzyme in the complex) although they were still insuMciently narrow to allow coupling constants to be measured. The T-phosphate signal showed no change in shift on forming the ternary complexes and is independent of pH in the range 5-7. One pyrophosphate phosphorus has a large upfield shift (2.23 p.p.m.) which is the same in both ternary complexes while the other experiences different upfield shifts (1.75 p.p.m, for folate and 2.17 p.p.m, for methotrexate) in the two complexes.

Discussion 2' Phosphate 9roup The 31 p chemical shift of the 2'-phosphate 31p nucleus has essentially the same value ( + l . 1 7 p.p.m.) in the complexes of N A D P H and N A D P + with the E. coil dihydrofolate reductase and the shift remains unchanged

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31p n.m.r, studies of complexes of N A D P H and N A D P ; : P. J. Cayley et al.

in the ternary complexes with the substrate analogues. This clearly indicates that the 2' phosphate group is binding in the same ionization state and to the same binding site on the enzyme in all the complexes. The observed chemical shift of the bound 2' phosphate group ( + 1.17 p.p.m.) is close to the value for this group in its dianionic state in the free coenzymes ( + 0.92 p.p.m, for NADPH). It is thus very probable that the 2' phosphate group is binding in its dianionic state to the enzyme. A similar conclusion was reached previously for the coenzyme complexes with the L. casei enzyme 5 7. As in the L. casei enzyme coenzyme complexes, the 2'-phosphate chemical shift is pH-independent in the range 5 7, where large titration shifts are observed for the free coenzymes (see Figure 2). The pK of the 2' phosphate group has been decreased by at least 2 pK units on binding, which points to this group being involved in strong electrostatic interactions with cationic group(s) on the enzyme. The 2'phosphate signal in bound N A D P H is 0.25 p.p.m, to low field of its value in free dianionic N A D P H , whereas a much larger extra downfield shift (1.7 p.p.m.) is observed in the complex with the L. caseienzyme. These shifts could arise from changes in hybridization of the phosphorus atom accompanying small distortions of the O P O bond angles ~ when the phosphate oxygens bind to the enzyme. (Changes in the ~ torsion angles are also related to O - P O bond angle distortion~2.) Recent studies of the X-ray structure of the ternary complex of the L. casei enzyme with N A D P H and methotrexate have confirmed that there are two cationic groups on the enzyme, Arg 43 and His 64, close to the 2' phosphate group. In fact, Matthews et al. ~ have suggested that the charged imidazole ring of His 64 is interacting directly with the 2' phosphate group. This residue is not conserved in the E. coli enzyme and it is possible that the absence of the extra bound shift reflects the absence of this His 64-2'phosphate interaction. Clearly some other cationic residue (possibly the conserved Arg 43) must be binding to the 2' phosphate in order to stabilize its dianionic state in the E. coli enzyme complex. The additional interaction from the imidazole group of His 64 in the L, casei enzyme complex could easily result in a small distortion of the O P O bond angles and thus lead to the observed extra bound shift. The absence of the His 64 interaction with the 2' phosphate group in E. coli enzyme complexes would help to explain the lower binding constant of N A D P H to the E. coli enzyme compared with that to L. casei. His 64 could also be irfiplicated in the decrease in N A D P H binding to the L. casei enzyme at pH values >6.5 (Ref 13), whereas the binding of N A D P H to the E. coli enzyme is independent of pH in this range ~4. In the single resonance 3~p spectrum of the N A D P H complex with E. coli dihydrofolate reductase (Figure 1c) the 2'-phosphate resonance is a 7.8 Hz doublet due to spin spin coupling between the 3~p nucleus and the 2' proton. This three-bond coupling can be related to the dihedral angle about the O - C 2, bond in the bound state using a Karplus-type curve proposed by Smith and coworkers ~5. Because of the strong electrostatic interactions between the dianionic phosphate group and residues on the enzyme, we can confidently assume that in the complex the phosphate group is in a fixed conformation. A 3JHp value of 7.8 Hz corresponds to one of the four possible dihedral angles: 0 = 3 2 , 1 2 4 , 2 3 4 and 328 . The 0 = 1 2 4 , 234 conformations have the O P2 bond

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Int. J. Biol. Macromol., 1980, Vol 2, August

almost eclipsed with the C2 C~ or C 2 C3 bonds and would be energetically unfavourable. The most likely conformations are therefore those with 0 = 3 2 ( + 10) or 328 ~ (_+ 10) as shown below: H

H

0=32

0=328

The doublet splitting observed on the 2'-phosphate signal of the N A D P H complex with L. casei dihydrofolate reductase (Figure ld) is very similar in magnitude (Jell = 7.5 Hz). The bound conformation of the 2' phosphate group is thus essentially the same as in the E. coli enzyme complex, despite the differing interactions of the dianionic 2' phosphate group with charged residues on the E. coli and L. casei enzyme. Similar coupling constants are also observed in the spectra of the ternary complexes of the enzymes with N A D P H and methotrexate indicating that the 2' phosphate has the same conformation as found in the binary complexes. The conformation of the 2' phosphate group in N A D P H in the ternary complex with methotrexate and the L. casei enzyme in the crystal state has been reported recently by Matthews and coworkers 1. The dihedral angle for the O - C 2 bond is 0 = 331 ° which agrees very well with one of the two possible values ( 0 = 3 2 8 + 10 °) found in solution. Pyrophosphate groups In all the complexes studied, the two pyrophosphate 31 p nuclei are shielded differently, indicating that they are influenced differently by binding to the enzyme. The resonances are always substantially to higher field of their values in the free coenzymes and they are also to higher fields (by 0.7 1.7 p.p.m.) of the signals in the corresponding complexes with L. casei dihydrofolate reductase. The py?ophosphate signals have not yet been assigned unequivocally; however, from a comparison of the X-ray crystallographic and 3~P-1H spin coupling constant results for the L. casei-methotrexate-NADPH complex it seems likely that the low field signal arises from the nicotinamide pyrophosphate 31p nucleus16. For the E. coli enzyme complexes where single resonance 31p spectra have been obtained, as for the complexes with the L. casei enzyme 5'6, it is always the low field signal which shows the 3 1 p O - C 1H 5 coupling. This suggests that the low field signal (A) arises from the nicotinamide pyrophosphate phosphorus in all the complexes reported in Table 2. Both the pyrophosphate resonances show larger changes in chemical shift on binding to the E. coli enzyme than on binding to the L. caseienzyme [see Table 2). These

Table 2 The 31p chemical shift changes (p.p.m.t of the pyrophosphate 3~p nuclei of NADPH on binding to E. coil and L. ca.sei dihydrofolate reductase

P (nicotinamide) P ladeninet

E. <.oli

L. casei

1.82 - 3.73

-0.16 - 2.69

3 1 p n.m.r, studies of complexes of N A D P H and NADP+ : P. J. Cayley et al.

shift differences probably reflect slightly different distortions of the O - P - O bonds in the two cases with the E. coli enzyme complexes having the larger disto'rtions. (Although increased distortion can be correlated with increased changes in 3 t p shifts, the latter cannot be directly related to the interaction energies.) There are clearly substantial differences in the observed 31p chemical shifts for the pyrophosphate nuclei in the various complexes (compare the 31p shifts in Table 1 of N A D P H and N A D P + in the ternary complexes with methotrexate and the enzyme). However, the overall pattern of upfield shifts is the same in all cases. Based on Gorenstein's empirical correlation of 3 1 p shifts with O P O bond angles it is evident that quite minor changes in angle ( < 1'') could give the chemical shift variations seen in these complexes. Such changes in O P O bond angles could easily result from differences in the interactions between the pyrophosphate oxygen atoms and the neighbouring protein residues in the different complexes. When N A D P H is bound to the E. eoli enzyme the high field 3 1 p signal of its pyrophosphate group shows no evidence of 31p_o._C_lH spin coupling (Figure l c). This implies either that the relaxation rates of the coupled 5'CH 2 protons are sufficiently fast to remove the spin spin interactions or, more likely, that the O - P bond is gauche to both H5' protons, in which case the Couplings would be expected to be very small ( < 5 Hz), as observed previously for the complex with the L. casei enzyme. This is the conformation found for free NADPH. The low field pyrophosphate signal shows unresolved 3 1 p - o C - I H splittings corresponding to a value of at least 8 + 2 Hz for the sum of the vicinal coupling constants (Jp o c Hs.q-Jp o c ,~). This indicates, from the Karplus equation of Smith and coworkers 15 that the conformation about this 5'C-O bond (probably that of the nicotinamide moiety) has changed from the gauche-gauche conformation by at least 30. This result is somewhat different from that found in the L. casei complex where the observed coupling constants (J,~p o c H~ -k-J.,,e o c n~>/ 16+5 HZ) are larger and indicate a change in conformation from the gauche gauche position ofat least 50. It is not possible to decide if these results represent a real conformational difference between the two complexes or simply that the 5' methylene protons are relaxed more efficiently in the E. eoli enzyme complex. It is worth noting that the X-ray crystallographic results on the complex of the L. easel enzyme with N A D P H and MTX ~'16 confirm quantitatively our original findings 5 on the conformation of the bound coenzyme in solution, suggesting that in this case relaxation effects are unimportant.

Conclusions 31p n.m.r, measurements on the E. coli enzymecoenzyme complexes provide the following information. (a) The 2' phosphate group binds in the dianionic state, interacting strongly with some cationic residue(s) on the enzyme in all complexes studied. Similar behaviour has The

been seen previously for the L. casei enzyme coenzyme complexes, although in these the presence of an interaction between the 2' phosphate and His 64 appears to cause a larger distortion of the O P O bond angle and probably contributes significantly to the 100-fold stronger binding of N A D P H to the L. casei enzyme. The P O - C 2 - H 2, dihedral angle ( ~ 33ff~) is essentially the same in the complexes with the E. eoli and L. casei complexes and agrees well with the value measured in the crystal studies on the L. casei enzyme N A D P H - M T X complex. (b) The pyrophosphate resonances show evidence for increased O P - O bond angle distortion compared with that in the complexes with L. casei enzyme. However, the change in the P - O - C s - H 5, torsion angle for the nicotinamide pyrophosphate is considerably less than was observed for the L. casei enzyme complex. The environment of the pyrophosphate group clearly differs in detail in the two Complexes, presumably because not all the residues with which it interacts are conserved.

Acknowledgements The trimethoprim-resistant strain E. eoli B RT500 was kindly provided by Dr J. J. Burchall (Burroughs Wellcome Research Laboratories, Research Triangle Park, North Carolina). We would also like to thank Dr G. C. K. Roberts for helpful discussions.

References 1

2 3 4 5 6

7 8 9 l0 11 12 13 14 15 16

Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S. T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N. and Krout, J. J. Biol. Chem. 1978, 253, (19), 6946 Hitchings, G. H. and Burchall, J. J. Adv. Enzymol. Relat. Sul~j. Biochem. 1965, 27, 417 Blakley, R. L. 'The Biochemistry of Folic Acid and Related Pteridines', Amsterdam, North-Holland Publishing Co. 1969 Way, J. L.,.Birdsall, B., Feeney, J., Roberts, G. C. K. and Burgen, A. S. V. Biochemistry 1975, 14, 3470 Feeney, J., Birdsall, B., Roberts, G. C. K. and Burgen, A. S. V. Nature {London) 1975, 257, 564 Feeney, J., Birdsall, B., Roberts, G. C. K. and Burgen, A. S. V. in "N.m.r. in Biology" (Eds. R. A. Dwek, 1. D. Campbell, R. E. Richards and R. J. P. Williams) Academic Press, London, 1977, p. 111 Birdsall, B., Roberts, G. C. K., Feeney, J. and Burgen, A. S. V. FEBS Lett. 1977, 80, 313 Baccanari, D. P., Averett, D., Briggs, C. and Burchall, J. Biochemistry 1977, 16, 3566 Gorentstein, D.G.,Kar, D.,Luxon, B.A. andMomii, R . K . J . Am. Chem. Soc. 1976, 98, 2308 Scudder, P., King, R. W. and Cayley, P. J. unpublished results Gorenstein, D. G. J. Am. Chem. Soc. 1975, 97, 898 Gorenstein, D. G. and Kar, D. Biochem. Biophys. Res. Commun. 1975, 65, 1073 Dunn, S. M. J., Batchelor, J. G. and King, R. W. Biochemistry 1978, 17, 2356 Cayley, P. J. and Dunn, S. M. J. unpublished results Blackburn, B. J., Lapper, R. D. and Smith, I. C. P. J. Am. Chem. Soc. 1973, 95, 2873 Manhews, D. A. personal communication

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