Structure-activity relationships in papain and bromelain ligand interactions

ARCHIVES

OF

BIOCHEMISTRY

Structure-Activity

AND

BIOPHYSICS

183,

38%3%

(1977)

Relationships in Papain and Bromelain Interactions’

Ligand

CORWIN HANSCH, R. NELSON SMITH, ARLEEN ROCKOFF, DANIEL F. CALEF, PRISCILLA Y. C. JOW, AND JAMES Y. FUKUNAGA Department

of Chemistry,

Pomona College, Claremont,

Received

February

California

91711

7, 1977

for 16 methyl hippurates The parameters K,,z and k,,, were determined (CH,OCOCH,NHCOC,H,-Xl hydrolyzed by papain. A simple linear relationship is found between log UK,,, and the hydrophobic substituent constant V. It is found that log k,,, is parabolically related to rTT.The results with papain are compared with results obtained by Hawkins and Williams with the enzyme bromelain. The two enzymes behave in a similar fashion.

studies in which the detailed structure of the enzyme is not known (1,2) but we have recently directed our attention to enzymes whose complete structures are known (3, 4). The purpose of this report is to extend our earlier study of ligand interactions with papain, an enzyme whose structure has been well worked out (Fig. 1). It is hoped that by comparing the amino acid composition of the binding areas with correlations by n or MR, techniques can be developed to characterize various binding regions of enzymes without using highly purified enzyme. At present there is no rigorous deterministic way of exploring enzyme surfaces in solution or in viva. We believe that important information about the composition of an enzyme in solution can be obtained via an extrathermodynamic analysis (5) of the perturbations of a well-designed set of molecular probes. Eventually, it should be possible to confirm the results of such studies by means of crystallographic analysis of ligands bound to enzymes. The equation,

An important problem in understanding the binding of ligands to macromolecules is that of delineating the role of the relatively nonspecific forces. There is considerable confusion about these as authors sometimes speak of London dispersion forces and sometimes of hydrophobic binding without making a distinction between the role of each in binding processes. It has been evident with enzymes whose X-ray crystallographic structures have been worked out that, in a general way, apolar groups tend to form clumps within the enzyme and polar groups tend to be located so that they contact the surrounding aqueous medium; of course, there are mixed regions of both polar and apolar amino acids. However, using the phenomenological approach, we have obtained evidence that substituent groups on inhibitors, as well as substrates, show two distinct kinds of nonspecific interactions: one correlated with the hydrophobic constant rr and the other correlated with the molar refractivity (MR)’ of the substituent. Most of the evidence for the two types of interactions has been obtained from enzymic I This investigation was supported by U.S. Public Health Service Research Grant No. CA-11110 from the National Cancer Institute. This is the second paper in a series. Part I is Ref. (3). 2 Abbreviations used: MR, molar refractivity; QSAR, quantitative structure-activity relationship; mp, melting point.

log l/K,,, = 0.57MR + 0.56~ - 1.921 s + 3.74 z”o 0.;90 0.148, was formulated

in our first

[II

publication

383 Copyright All rights

0 19’77 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003-9861

HANSCH

384

FIG. 1. X-ray

structure

[ (3), from data of Williams et al. (6, 711on the quantitative structure-activity relationship (QSAR) of congeners of I and II binding to the enzyme papain: 0 @-.\

I xx.=/

dkHs

ET AL.

NHSOzCH3 T I

K,,, in Eq. [l] is the Michaelis constant, which was assumed to be the reciprocal of a binding constant for the various substrates. Care must be taken in making such an assumption since K,,< may contain information on kca,. For Eq. 111, n represents the number of data points on which the equation is based (13 congeners of I and 7 congeners of II), r is the correlation coefficient, s is the standard deviation from the regression, MR is the molar refractivity of X, and u is the Hammett constant for X. The indicator variable I takes the value of 1 for congeners of I and the value of 0 for congeners of II and is simply a device for merging two equations whose only significant difference is in their intercepts. The X-ray crystallographic structure of papain formulated by Dickerson and Geis (8) from the work of Drenth is shown in

of papain

Fig. 1. We have added X-Y to the picture to represent how substituents on congeners I, II, and III might contact enzymic space. It was concluded from Eq. [ 11that X of I and II binds to the bank of polar amino acids (Lys 17, Asn 18, Glu 19, Gly 20, Ser 21, Gly 23, Gly 62, Asp 64, Gly 54) situated near the catalytic jaws of Cys 25 and His 158. This conclusion was based on the fact that K,,, was correlated with MR rather than n. Such information indicates that the binding region on the enzyme for X is not hydrophobic. Positioning in this manner means that the moieties -NHSO,CH,, and -NHCOC,H, might fall in the crevice between the two hemispheres of the enzyme. The difference (9) between r for these two groups is 1.67. Assuming hydrophobic interaction for these two elements, we calculate (3) a slope of 1.15 for the dependence of log l/K,,, on 7~; i.e., (1.67) (slope) = 1.92 where 1.92 (the coefficient of TTin Eq. [l]) represents the average difference in log UK,,, for the two sets of congeners on which Eq. [ll is based. The purpose of the research reported in this paper was to make a set of congeners (III) which would presumably bind in the

CH30

1

O CH,NH!-.<

*y .=.

III papain crevice to determine if correlation with 7~would indeed result. Because of the

PAPAIN

AND

BROMELAIN

apolar character of many of the acids surrounding the crevice, as our calculation based on only two we anticipated that correlation would be found. EXPERIMENTAL

amino well as points, with 7~

PROCEDURES

Enzyme-substrate parameters. Papain, 2X crystallized and suspended in 0.05 M sodium acetate at pH 4.5, was obtained from Sigma Chemical Co. and kept refrigerated. For a given day’s work this concentrated solution was, in most cases, diluted with 1.25 x lo- 5 M 0.30 M NaCl to give an approximately stock solution which was activated, with stirring, with a tiny crystal (thiocresol is very insoluble in water) ofp-thiocresol for 2 h or more before use. The rate of enzymic hydrolysis of the ester substrates was followed by automatic titration. using a Radiometer pH-stat set at pH 6.00. Standardized millimolar NaOH was stored under N, and used as a titrant in a flowing N, (“prepurified” from Matheson Gas Co.) atmosphere. The temperature of the reaction vessel was maintained at 25.O”C with a thermostated circulating water jacket. A reaction mixture normally consisting of n ml of ester substrate of known concentration dissolved in 0.30 M NaCl, 1.000 - x ml of 0.30 M NaCl, and 0.100 ml of 0.10 M EDTA in 0.30 M NaCl was equilibrated at 25.O”C and adTABLE PARAMETERS

385

INTERACTIONS

justed to a pH of exactly 6.00 by automatic titration (this small volume of NaOH was recorded and ineluded in the concentration calculations). The NaCl and EDTA stock solutions were previously adjusted to a pH very close to 6.00. After selecting an appropriate time span for the recorder, enzymic hydrolysis was initiated by rapid addition of 0.100 ml of activated papain solution. The papain concentration in the reaction mixture was about 10 Ii M. In calculating the initial rates, a suitable correction was made for the reduction caused by progressive dilution resulting from addition of titrant. The slope of the corrected initial recorder trace was determined with a specially constructed protractor which could be read to the nearest 0.083”. These slopes, when converted to moles per minute, constituted the initial rates for the various initial substrate concentrations. In most cases, 6 to 12 different substrate concentrations were used to generate each computerized Lineweaver-Burk equation, using Cleland’s method (10) of weighted least squares. The values of K,,, and k,.;,, obtained from these Lineweaver-Burk equations are shown in Table I and were used in the structure-activity correlation Eqs. 131-151. Two methods were used for the synthesis of the hippurate esters (methyl glycinatesl: Method A. Acid chloride (X-C,H,COCll 0.05 mol was added to 150 ml of chloroform containing 0.07 mol of the methyl ester of glycine and 0.12 mol of I

OF EQS. 13]-151 FOR THE INTERACTION CH.,OCOCH.,NHCOC,H-Y WITH PAPAIN

USED IN THE FORMULATION

Y

4-NH, 4-NHCOCH, H 4-CN 3-NOp 3-OCH,, 3-CN 4-NO, 4-OCH,, 3,5-di-NO, 3-CH, 4-CH:, 4-Cl 4-t-C,H,, 4-I 4-O&H,,

LIGAND

n

-1.05 -0.63 0.00 ~0.16 0.13 0.21 -0.12 0.18 0.22 0.19 0.54 0.54 0.91 1.87 1.35 1.84

I’ From Ref. (91. ’ K,,, is in molar units. ’ k,.,, is in s’. ” Calculated from Eq. 131. ’ Calculated from Eq. 151.

MR”

0.54 1.49 0.10 0.63 0.74 0.79 0.63 0.74 0.79 1.48 0.56 0.56 0.60 1.96 1.39 2.17

log l/K,,,”

OF

log k Fat

Obsd

Calcd”

Obsd

Calcd’

0.506 0.850 1.27 1.38 1.42 1.47 1.61 1.47 1.63 1.66 2.02 2.04 2.41 3.10 3.10 3.45

0.404 0.826 1.46 1.30 1.59 1.67 1.34 1.64 1.68 1.65 2.00 2.00 2.37 3.34 2.82 3.31

0.336 0.453 0.534 0.652 0.930 0.111 0.760 0.348 0.013 -0.182 0.029 0.037 -0.128 -2.693 -1.482 -2.172

0.717 0.778 0.539 0.637 0.440 0.371 0.615 0.398 -0.362 -0.389 0.019 0.019 -0.505 -2.502 ~1.306 -2.426

386

HANSCH

triethylamine. The acid chloride was added slowly with good stirring because of the exothermic reaction. After 1 h of stirring, 250 ml of ether was added and the resulting precipitate of triethylamine hydrochloride was removed by filtration. The filtrate was vacuum-evaporated and residue-purified by recrystallization. Method B. A solution of the appropriate hippuric acid was refluxed for 2 h in methanol saturated with dry HCl. This mixture was then vacuum-evaporated, the residue was neutralized with bicarbonate solution, and the product was extracted with CHCl:,. After drying the CHCl,, over magnesium sulfate it was evaporated and the residue was crystallized One derivative, methyl hippurate, was a commercial sample of established purity. Methyl 4-nitrobenzoylglycinate (A) recrystallized from methanol-water, mp 156-157”. Anal. (C,,,Hu,N,O,) C, H. Methyl 4-methylbenzoylglycinate (A) recrystallized from methanol-water, mp 92-94”. Anal. (C,,H,,NO,,) C, H. Methyl 4-methoxybenzoylglycinate (A) recrystallized from benzene, mp 110-115”. Anal. (C,,H,:,NO,) C, H. Methyl 4-iodobenzoylglycinate (A) recrystallized from dimethylformamide-water, mp 115-117”. Anal. (C,,,H,,,NO:J C, H. Methyl 4-cyanobenzoylglycinate (A) recrystallized from dimethylformamide-water, mp 178180”. Anal. (C,,H,,,N,O,) C, H. Methyl 4-t-butylbenzoylglycinate (A) recrystallized from methanol-water and dimethylformamide-water, mp 92-93”. Anal. (C,,H,,NO,l C, H. Methyl 4-butoxybenzoylglycinate (A) purified by chromatography on silica, mp 91-92.5”. Anal (C,,H,!,NO,) C, H. Methyl 3-nitrobenzoylglycinate (A) recrystallized from methanol, mp 101.5-102.5”. Anal. (C,,,H,,,N,O,) C, H. Methyl 3-methoxybenzoylglycinate (A) recrystallized from methanol-water, mp 77-79”. Anal. (C,,H,,,NO,) C, H. Methyl 3-methylbenzoylglycinate (A). This product remained liquid even after purification on a silica column, bp -210”/0.4 mm. Anal. (C,,H,,NOJ C, H. Methyl 3-cyanobenzoylglycinate (A) crystallized 93.5-95.5”. Anal. from methanol-water, mp (C,,H,,,NO,) C, H. Methyl 4-acetylaminobenzoylglycinate (A) prepared by acetylation of the 4-aminoglycinate; recrystallized from dimethylformamide-water, mp 204-206”. Anal. (C,,H,,N,OJ C, H. Methyl 3,5-dinitrobenzoylglycinate (Ai crystallized from chloroform-ether, mp 128.5-130”. Anal. (C,,,H,,N:,O;) C, H. Methyl 4-aminobenzoylglycinate (B) crystallized

ET AL. from methanol-water, mp 129-131.5”. Anal. (C,,,H,,N,O:,) C, H. Methyl 4chlorobenzoylglycinate (B), mp 118.5119.5”. Anal. (C,,,H,,,ClNO,,) C, H. The purity of all compounds was checked by thinlayer chromatography. All elemental analyses agree with the theoretical value within 0.35. Substituent cor~stants. The MR and (r constants were taken from our recent compilation. To simplify matters, 7~ constants from benzamides were employed since a fair number of these have been determined (11). T, = logP,,~‘,,,,cw,

~ l%P, ,11;u1\111~

where P is the octanoliwater partition coefficient (12). Several new x constants for the benzamide system were calculated from the following new 1ogP values for benzamides using log P of 0.64 for benzamide itself: Log P

X-Benzamide

2.48 0.01 2.51 0.48 0.52 1.99 0.85 0.83 -0.41

4-O&H<, 4-NHCOCH,, 4-t-C,H,, 4-CN 3-CN 4-I 3-OCH:, 3,5-di-NO, 4-NH,

t ? 2 -t i * + i-c

77

0.01 0.02 0.03 0.01 0.02 0.13 0.01 0.02 0.01

1.84 0.63 1.87 -0.16 -0.12 1.35 0.21 0.19 -1.05:’

In order to establish that r values from benzamide are indeed close to n values from X-benzoylglycinates, log P values were measured for three of the esters: methyl benzoylglycinate = 0.83, methyl 4= 0.98, 4-aminobenzoylglynitrobenzoylglycinate cinate = -0.23. The r values of n&110:, = 0.16 and error, the TG-UH2= -1.05 are within experimental same as those in Table I from the benzamide system. The 4-NO, and 4-NH, substituents were selected since it is known that the electronic effect of substituents is the most important parameter affecting the variability of T from system to system (13). Hence, the strong electron-withdrawing NO, and the strong electron-releasing NH, groups should show the largest variation in TTvalues between the two systems, benzamide and methyl hippurate. There is a close relationship between r values from the benzene system (9) and those from the benzamide system as shown in the following correlation equation: ~h.wmlKl<. = O.&39n,,,.,,,,.,,,, + 0.183~ + 0.237 n r s 29 0.985 0.133. :I Our previously error (11).

reported

value

for 4-NH,

is in

PAPAIN

AND

BROMELAIN

LIGAND

INTERACTIONS

387

strate complex. An alternative explanation is that large hydrophobic groups may We have formulated the equation, produce a conformational change in the enzyme and thereby render it less effective log l/K,,, = 1.005(~0.11)7T + l.4a59(?o.lo) [31 as a catalyst. Equation [5] is a more significant correlation than Eq. 141 (F,, 1:1= 17.7; F,, 1:gu.fj,jl 1: O.&l 0.165, = 17.8) and brings out the optimum degree from the data in Table I via the method of of hydrophobicity needed for the hydrolyleast squares. The figures in parentheses sis step. Setting the derivative of Eq. 151 in this expression are the 95% confidence equal to 0, we calculated an optimum limits. Using MR instead of x in Eq. [3] value of 7~ of -0.69 (-2.05 to -0.23); TT gives a much poorer correlation (r = 0.615) should be negative for maximum activity and the addition of a term in u, MR, or 7~~ in the catalytic step. does not significantly improve the correlaSince the publication of the drawing of tion. Since 7~ and MR are reasonably or- Fig. 1, an additional amino acid has been thogonal vectors (9 = 0.361, as is also true shown to be in the sequence which in this for Eq. [l], Eq. [3] establishes two disdrawing is valine between 129 and 130. tinctly different types of enzymic space. This insertion undoubtedly has some effect We were pleased to find a slope of 1.00 on the overall geometry but, for our apfor Eq. [3], near that which we had pre- proximation, does not alter our original dicted from two data points. It is also of conclusions. interest to note that the 4-OCH,Looking at Fig. 1, if we divide the active CH,CH,CH, group is well fit; this supports site cleft through the catalytic groups (Cys what one might surmise from the size of 25 and His 158 in this drawing), the upper the crevice in Fig. 1, that quite large portion residues are mostly polar: Asn 18, groups (i.e., CH,NHCOC,H,OC,H,) could Gln 19, Gly 20, Ser 21, Cys 22, Gly 23, Ser be accommodated in this space. 24, Asn 64, Gly 65. The residues in the In the case of the congeners of III, the lower portion are mostly nonpolar: Ala 12, nature of Y has a large effect on k,.,, and Val 13, Thr 14, Pro 15, Val 16, Trp 26, Ser the following QSAR show how this relates 29, Val 32, Thr 33, Gly 66, Tyr 67, Pro 68, to the hydrophobicity of Y: Trp 69, Vall30 (not in Fig. l), Ser 130, Val 132, His 158, Ala 159, Va1169, Ala 161, Ala + 0.375(?0.31) 1% %a, = -1.239(,0.371n 162. In Fig. 2, each of these residues has a [41 number one higher than in Fig. 1; e.g., Ser 1”s 0.8’89 0.5s28, 130 in Fig. 1 is Ser 131 in Figs. 2 and 3. log k,,, = -0.499( 50.2637; - 0.693( 20.37) 7J This way of looking at the active site is + 0.5391 -tO.23) [51 good for a general view but it is obviously necessary to consider the orientations of ;“s 0.9r55 0.3s57. amino acid side chains; a tryptophan oriEquation 141is not nearly as good a correented away from the active site pocket lation as Eq. [31. The slope of Eq. [4] is would not contribute to the hydrophobicity about the same as Eq. [3] except that it is of the environment any more than a glynegative, which indicates a diametrically tine. The location of the side chains shapes opposed hydrophobic force inhibiting the the binding site; for this reason, a more catalytic step. Such a result is not unexdetailed study of the protein was made pected as we have observed it before in the using coordinates which were kindly suphydrolysis of X-phenylglucosides by emulplied to us by Professor Drenth (16). Figure 2 is a stereo view of the active sin (14, 15). One rationalization of this site region looking down on the “top” of the effect is that hydrophobic substituents hinder desorption of the acid portion of enzyme as seen in Fig. 1. Only the significant side chains are drawn; the rest of the the hydrolyzed ester, preventing another molecule from forming the enzyme sub- region around the active site is shown as RESULTS

AND

DISCUSSION

388

HANSCH

FIG. 2. Stereo view of the region view as Fig. 1.

FIG.

ET AL

around the active

site of papain

3. Stereo view of the active site of papain at right

lines connecting the a-carbons. The picture is of course of crystalline enzyme without a bound ligand. The catalytic Cys 25 and His 159 are in the middle with the polar bank on the left side of the catalytic groups consisting of residues 19 to 24 and possibly 63 and 64, although they seem to be slightly further removed and somewhat shielded by residue 23. The disulfide bridge between Cys 22 and Cys 63 (not

from the same point of

angles to that of Figs. 1 and 2.

shown in Fig, 2) would seem to stabilize this region. The hydrophobic region is harder to characterize. Figure 2 shows the depth of the cleft as seen in Fig. 1 but gives a more crowded, less open picture. Atoms from residues Ser 29 and Ala 160 would seem to block the center of the cleft to large ligands; this can also be seen in Fig. 3, which is a view into the cleft at a right

PAPAIN

AND

BROMELAIN

angle to Fig. 1. Pictures sent to us by Professor Drenth show the enzyme with a bound carbobenzoxy-phenylalanyl-glycine derivative with the benzyl moiety of the phenylalanine residue resting roughly between residues Tyr 67 and Val 133. Major shifts of the residues around the active site do not seem to occur on the binding of this ligand. If we assume the hippurates in our study bind similarly, then the hydrophobic region is a pocket on one side of the cleft consisting of hydrophobic residues Tyr 67, Pro 68, Trp 69, Ala 160, Val 133, and Tyr 207. In addition, there are several serines in the region: 29, 132, and 206. Certain nonpolar amino acids Trp 26, Val 132, and Val 161 have their side chains oriented away from the cleft, and hence, would not seem to contribute to its hydrophobic character. Figure 3 gives another view looking directly into the cleft. It must be kept in mind that the enzyme is not as rigid as the pictures suggest. It is possible that, through a kind of “breaththe hippurates ing” action in solution, could bind deep in the cleft back beyond residue 29, as shown in Fig. 3. It is the polar region just outside of the crevice to which X of I and II appears to bind. This binding is correlated with MR of X and is favored by electron withdrawal by X. One would expect the polar side chains of the amino acids in this region which is open to the aqueous phase to be strongly solvated by water. It is also expected that the polar functions of X (OH, OMe, CHO, COMe, and NO,) are well solvated while the nonpolar groups such as F, Cl, and CH, would not be so strongly solvated. The fact that log l/K,,, is not correlated by 7~ for X of I and II suggests that desolvation is not occurring and that dispersion forces and dipolar interactions must account for differences in log UK,,, . It is also found that adding a term in 7~to Eq. [ll does not improve the correlation. One is left with the feeling that X with its solvent shell somehow freezes to the solvated surface of polar amino acids. Felix Franks (17) has gathered evidence for a second type of “hydrophobic” interaction between small molecules in aqueous solution in which the solvent shells appear not to be removed when two groups of

LIGAND

INTERACTIONS

389

atoms come together; instead, they seem to reinforce their structures and “freeze” together. This mechanism of binding of polar and apolar substituents onto polar enzymic space is attractive because it offers a way to explain the lack of importance of hydrogen bonding and specific dipole-dipole interactions as well as specific polarization effects along preferred bond axes (18). It would be expected that any such strong specific effects would prevent good correlation with MR. Equation [ll is based on the following variations of X in the 3- and 4-positions of congeners of I and II; H, F, Cl, NHP, OH, CH:I, OCH:<, CHO, COCH:l, NO,. Water of solvation around these various groups could greatly attenuate the large differences in geometry, dipole moments, and hydrogen-bonding abilities. The very high correlation coefficient with Eq. 111is in part an artifact of merging two data sets rather widely separated in substituent space. This combining produces a great increase in total variance in the data. Breaking Eq. Ill into two equations (3) gives one equation based on 13 congeners of I with r’ = 0.874 and another equation based on 7 congeners of II with r’ = 0.943. Hence, o and MR account for about 90% of the variance of a quite diverse set of substituents. The 10% “unexplained” variance is in part due to experimental error in K,,,, u, and MR and in part to unaccounted interactions such as hydrogen bonding, dipolar interactions, and specific polarization along certain bonds. Experimental error in the constants must be on the order of 3 to 5%, which leaves very little information unaccounted for. It is indeed surprising to us that such good correlations can be found for enzyme ligand interactions (1, 2) with such a general measure of dispersion forces as MR. Pauling and Pressman (19) appear to be the first to have attempted to use MR in correlating binding of micromolecules with macromolecules. The exact meaning of MR in equations such as Eq. [l] remains a mystery and needs more attention by theoreticians. Equation 131is simpler to understand as it contains only one term which “explains” 96% of the variance in binding of 16 quite

390

HANSCH

different analogs. The coefficient of near 1 with 7~ brings out the close parallel between binding to papain and partitioning into octanol. Since such a geometrically diverse set of substituents is so well correlated by octanollwater 7~ values, there must be a large amount of flexibility in the hydrophobic pocket. Binding here is not of the “lock and key” type. The results with papain support the hypothesis that one can, in a limited way, diagnose enzymic space by means of the parameters r and MR. In a recent report, Hawkins and Williams (20) give results on the hydrolysis of esters I and II by two forms of the enzyme bromelain. Only values for ho/K,,) -are given. Equations [6]-[S] have been formulated from the results in Tables II and III: Bromelain

B Hydrolysis

log k,,/K,,

6H 6

= 0.505( k0.34)MR

+ 0.653( kO.23)~ + 2.605( kO.21) n

r

s

10

0.961

0.125

TABLE PARAMETERS

USED IN THE FORMULATION

X-C,H,OCOCH,NHCOC,H, X

MR”

Bromelain D Hydrolysis of X-C,H,OCOCH,NHCOC,H, log k,,/K,,, = 0.460( k0.12)MR + 0.635(?0.09)a

+ 2.219

n

9 Bromelain

0.;95

B Hydrolysis

X-C H O!CH log k,,;K:

o.os41 of

NHSO CH,

= 0.46%(+0.2;)Mk

+ 0.526( +O.l3)at

0.;91

ISI

+ 1.131( tO.lO) 0.;63

CT”

II OF EQUATIONS BY BROMELAIN

FOR THE HYDROLYSIS B AND D

B”

B’

OF

D”

D”

log k ,lK,,, 4-NO, 4-OCH, 4-Cl 4-COCH,, 3-NO, H 4-F 3-a 4-NH, 4-CH, I’ From Ref. b From Ref. ’ Calculated (’ Calculated ( This data

0.74 0.79 0.60 1.12 0.74 0.10 0.09 0.60 0.54 0.56

]71

One data point (3-NO,) has been omitted in the formulation of Eq. [71; for some unknown reason it is very poorly fit (mispredieted by 10 standard deviations). It was observed in both Eqs. [6] and [7] that o gave a better correlation than (TV as was also true with Eq. Ill. In the same equa[61 tions, MR gave a much better correlation than Z-. The vectors n and MR are quite orthogonal for the set of substituents involved in these correlations. These results closely parallel those obtained with papain

of

X-C 6H 5-O!CH 2NHCOC

ET AL

0.78 -0.27 0.23 0.50 0.71 0.00 0.06 0.37 -0.66 -0.17

(9); MR values scaled by 0.1. (20). using Eq. IS]. using Eq. 171. point not used in deriving Eq. 171.

Obsd

Calcd

Obsd

Calcd

3.66 2.81 3.10 3.39 3.38 2.71 2.54 3.07 2.42 2.95

3.49 2.83 3.06 3.50 3.44 2.65 2.69 3.15 2.45 2.78

3.05 2.41 2.61 3.04 2.55’ 2.31 2.24 2.77 2.03 2.41

3.05 2.41 2.64 3.05 3.01 2.27 2.30 2.73 2.05 2.37

PAPAIN TABLE PARAMETERS

AND

BROMELAIN

III

USED IN THE FORMULATION

OF EQ. 181

FOR THE HYDROLYSIS OF X-C,H,OCOCHLNHSO,CH:, BY BROMELAIN B

X

4-NO, 4-OCH:, 4-CHO 4-Cl 4-COCH, 3-NO, H 4-F 4-OH

MR

W

0.74 0.79 0.69 0.60 1.12 0.74 0.10 0.09 0.28

log k,,lK,,,

1.24 -0.16 1.03 0.27 0.87 0.71 0.00 0.05 -0.16

Obsd”

Calcd”

2.18 1.45 2.00 1.57 2.10 1.75 1.25 1.14 1.15

2.03 1.48 1.80 1.64 2.11 1.99 1.20 1.23 1.11

” From Ref. (20). h Calculated using Eq. [81.

(3) and suggest that X is binding in polar space in bromelain as it does in papain. In the case of the mesyl derivatives of Eq. [8], it is found that (T- gives a better correlation than CT[r = 0.991 vs r = 0.946). The coefficients with the o term of Eqs. [6]-[8] are about the same. The better fit with (T- may be an artifact which a larger set of data would not support. The coefficient with (T- in Eq. [81 is not as large as one generally finds for typical reactions where the role of electron withdrawal is critical for the leaving phenoxide ion. Using 7~ instead of MR in Eqs. [61-[81 results in much poorer correlations. MR is a consistently better parameter than 7~in the two cases with papain (3) and in three cases with bromelain. Professor Henry S. Frank called our attention to the fact that the molar volume (MWId) in the Lorentz-Lorenz equation, MR

n” - 1 = _____.~~-~-

n”+2

MW d’

LIGAND

When MR was used, a 3 to 10% greater reduction in variance was found. Although for the data at hand MR and MWId are rather highly collinear, the results do suggest that more than simple volume of the substituents is involved; polarizability of the substituent electrons as modeled by n is also important. The above correlation equations for papain allow us to make an educated guess as to how other ligands bind. For example, Anderson and Vasini (21) studied the irreversible inactivation of papain by a set of N-alkylmaleimides. Table IV contains log k values for the apparent second-order rate constant for the inactivation of papain and MR and n values for the substituents; Eqs. [lo] and [ 111 have been derived from this: log k = 0.615(iO.l3)MR

is such a dominant factor in the equation because variation in n (refractive index) is relatively small (general range, 1.35 to 1.65). It seemed possible that MWId might correlate the data as well as MR; we therefore calculated MW/d from density and the refractivity index measurement taken from the literature. In five equations, two for papain (3) and three for bromelain, employing MWId in place of MR, gave poorer correlations in every instance.

+ 1.290(?0.411 I101

n 9

0.;74

0.1’80,

log k = 0.551(~0.12k~

+ 1.456(-tO.38) Llll

n 9

0.;74

O.l”sl.

The qualitative correlation with the two parameters is the same since the two vectors are almost perfectly collinear (r2 = TABLE

IV

PARAMETERS USED IN THE FORMULATION EQUATIONS FOR INTERACTION OF

OF

HCP

I-X

ii,

CHC %I WITH

PI

391

INTERACTIONS

X

Ethyl Butyl Pentyl Hexyl Heptyl octy1 Nonyl Decyl Phenyl

PAPAIN

log K Obsd”

Calcd’

2.22 2.41 2.61 2.86 3.48 3.72 3.91 4.33 2.70

1.92 2.50 2.78 3.07 3.35 3.64 3.92 4.21 2.85

MR

7i

1.03 1.96 2.42 2.89 3.35 3.82 4.28 4.75 2.54

1.0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 1.96

I’ Second-order inhibition constant from Ref. (21). ’ Calculated using Eq. [ 101.

392

HANSCH

0.97). However, the coefficient with MR in Eq. 1101is close to that with MR in Eq. 111, while the slope of Eq. Ill] is not at all close to that of Eq. [31; hence, we presume that the maleimides react irreversibly with the Cys 25 in such a way that the alkyl groups fall into the same space as X in Fig. 1. In summary, one can say that correlation analysis can be a valuable tool in delineating the parameters of interaction between enzymes and small organic compounds. When coupled with X-ray crystallography, it will greatly increase our understanding of enzyme-ligand interactions. REFERENCES C., AND HANSCH, C. (1975) J. Chem. Sot. 97, 6849-6861. 2. YOSHIMOTO, M., AND HANSCH, C. (1976) Chem. 19, 71-98. 3. HANSCH, C., AND CALEF, D. F. (1976) Chem. 41, 1240-1243. 4. YOSHIMOTO, M., AND HANSCH, C. (1976) 1. SILIPO,

Chem. 5. LEFFLER,

41, 2269-2273. J. E., AND GRUNWALD,

Amer. J. Med.

J. Org. J. Org.

E. (1963) Rates and Equilibria of Organic Reactions, Chap. 6, Wiley, New York. A. (1965)Biochem. J. 6. LOWE, G., AND WILLIAMS, 96, 199-204. 7. WILLIAMS, A., LUCAS, E. C., AND RIMMER, A. R.

ET AL (197215. Chem. Sot. Perkin Trans. II 621-633. R. E., AND GEIS, I. (1974) The Structure and Action of Proteins, p. 86, Harper and Row, New York. 9. HANSCH, C., LEO, A., UNGE:R, S. H., KIM, K. H., NIKAITANI, D., AND LIEN, E. d. (19731 J. Md. Chem. 16, 1207-1216. 10. CLELAND, W. W. (1967)Aduan. Enzymol. 29, l32. 11. HANSCH, C.. KIM, K. H., AND SARMA, R. H. (1973) J. Amer. Chem. Sot. 95, 6447-6449. 12. LEO, A., HANSCH, C., AND ELKINS, D. (19711 Chem. Rec. 71, 525-616. 13. FUJITA, T., IWASA, J., AND HANSCH, C. (1964) J. Amer. Chem. Six. 86, 5175-5180. 14. HANSCH, C., DEUTSCH, E. W., AND SMITH, R. N. (1965) J. Amer. Chem. Sot. 87, 2738-2742. 15. SMITH, R. N., POINDEXTER, T. P., AND HANSCH, C. (1975) Physrol. Chem. Phys. 7, 423-436. 16. DRENTH, J., JANSONIUS, J. N., KOEKOEK, R., SLUYTERMAN, L. A. A., AND WOLTHERS, B. G. (1970) Philos. Trans. Roy. Sot. London Ser. B 257, 231-236. 17. FRANKS, F. (1974) in Water (F. Franks, ed.). Vol. 4, Chap. 1, Plenum Press, New York. 18. INGOLD, C. D. (1969) Structure and Mechanism in Organic Chemistry, 2nd ed., p. 152, Cornell Univ. Press, Ithaca, N. Y. 19. PAULING, L., AND PRESSMAN, D. (1945) J. Amer. Chem. Sot. 67, 1003-1012. 20. HAWKINS, H. C., AND WILLIAMS, A. (1976) J. Chem. Sot. Perkin Trans. II 723-729. 21. ANDERSON, B. M., AND VASINI, E. C. (1970) Bzochemistry 9, 3348-3352. 8. DICKERSON,