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5 Chymotrypsinogen: X-Ray Structure
Chymotrypsinogen: X-Ray Structure J. KRAUT I. Introduction . . . . 11. The Activation Process . . 111. X-Ray Crystallographic Results A. Isoleucine 16 . . . B. Arginine 145 . . . C. Methionine 192 , D. Catalytic Site . . E. Activation Refolding .
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165 167 169 175 176 179 179 182
1. Introduction
The material to be covered here would logically have been incorporated into the chapter by Blow on chymotrypsin, instead of being presented as a relatively independent entity, except for the circumstance that the results described herein were still being prepared for publication while that chapter was being written. Therefore, sacrificing organizational elegance in the interest of currency, the authors of the two chapters have chosen to describe the three-dimensional structure of bovine chymotrypsinogen A and its relationship to the activation process separately. Throughout the rest of this chapter, bovine chymotrypsinogen A will be referred to simply as chymotrypsinogen or the zymogen. Almost all the material discussed here is based upon the paper by Freer et al. (I), wherein the preliminary results of the X-ray analysis of chymotrypsinogen at 2.5A resolution are presented. It is important to 1. S. T.Freer, J. Kraut, J. D. Robertus, H. T. Wright, and Ng. H. Xuong, chemistry 9, 1997 (1970). 166
BW-
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emphasize the word preliminary since experience has shown that protein structures determined by X-ray analysis have a decided tendency to undergo continuous minor modification over a period of years as small errors in the original model are discovered, electron density maps are gradually improved, errors in amino acid sequences are corrected, water molecules are discovered, and, most especially, as the investigators come to see subtle but significant features that may have escaped notice in the first burst of enthusiasm over a newly determined structure. I n the case of chymotrypsinogen, even as this is being written more careful comparison of the enzyme and zymogen structure is being carried out both in Cambridge and La Jolla, and improved electron density maps are being computed; thus, there is little doubt that the story presented here will soon prove to be incomplete. Nevertheless, some interesting and thought-provoking observations have already been made and will be summarized in this chapter. The phenomenon of induction of biological activity by limited proteolysis of an inactive precursor is fairly widespread in nature. It has been found in various forms in such widely separated types of organisms as bacteria, yeast, green plants, invertebrates, and vertebrates. The subject has been reviewed recently in detail by Ottesen (2), and so it need not be extensively surveyed here. The point to be made is that in a sense it represents a kind of biochemical amplification and control mechanism of which the conversion of chymotrypsinogen to chymotrypsin in the vertebrate duodenum, under the influence of the enzyme trypsin, is only one relatively simpIe though well-known example. Another much more complex example of limited proteolysis as a biological amplification and control mechanism is seen in the clotting blood. Here, a t least eight distinct factors interact in a cascading sequence that ultimately results in the conversion of soluble fibrinogen into an insoluble stabilized fibrin clot (3).A perhaps even more complex case familiar to the immunologist is that of the complement system. Enzymic cleavage is an essential feature of the activation mechanism of several complement components, of which 11 are now recognized ( 4 ) . Nor is the phenomenon of activation by limited proteolysis restricted to enzymes. The hormone insulin, which plays an important role in glucose metabolism, is now known to be the product of limited proteolysis of a single-chain proinsulin molecule. The latter is synthesized in specialized cells of the pancreas, where it is activated prior to secretion by the splitting out of a polypeptide connecting 2. M. Ottesen, Ann. Rev. Biochem. 36, 55 (1967). 3. E. W. Davie and 0. D. Ratnoff, in “The Proteins” (H. Neurath, ed.), 2nd ed., Vol. 3, p. 359. Academic Press, New York, 1965. 4. H . J. Muller-Eberhard, Ann. Rev. Biochem. 38, 389 (1969).
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the C-terminus of the insulin B chain with the N-terminus of the A chain (6). Thus, with the detailed architecture of the chymotrysinogen molecule now becoming clear, and the model of chymotrypsin itself already well established, we have our first glimpse of one simple example of this important type of biological phenomenon a t the level of molecular structure, II. The Activation Process
There are several steps involved in the process of activating chymotrypsinogen to give the various recognized forms of chymotrypsin. It is worthwhile considering what is known of this process and how the various chymotrypsins are related in order to help evaluate the significance of the structural differences, to be described below, between the zymogen and the a form of the active enzyme. A review of the chemistry of the activation process as it was understood in 1960 has been given by Desnuelle (6) in the previous edition of “The Enzymes.” Although Desnuelle’s review antedated the complete sequence determination, from the point of view of classical enzyme chemistry there is not very much that is new to be said about the subject; thus it will be only briefly summarized here. Figure 1 is reproduced from Wright et a2. (7).It is a suggested modification of the scheme generally accepted until now (6) but will nevertheless serve as a convenient summary of the nomenclature and chemical relationships among the forms of chymotrypsin. The proposed scheme differs from the classical one in that it supposes a-chymotrypsin to be conformationally different from 7-chymotrypin, and to be produced only by activation of the neo-chymotrypsinogen that has the dipeptide Thr 147-Asn 148 deleted, and not directly by degradation of S-chymotrypsin. Well-established features of the activation scheme are as follows: (1) Chymotrypsinogen is converted to the fully active n-chymotrypsin by trypsin catalyzed hydrolysis of the peptide bond between Arg 15 and Ile 16. It is this peptide-bond scission, and this one alone, that generates enzymic activity. All subsequent products result from autodegradation of the n-chymotrypsin molecule. (2) S-Chymotrypsin is missing the dipeptide Ser 14-Arg 15; S-chymo5. D. F. Steiner, J. L. Clark, C. Nolan, A. R. Rubenstein, E. Margobash, B. Aten, and P. E. Oyer, Recent Progr. Hormone Rea. 25, 207 (1969). 6. P. Desnuelle, “The Enzymes,” 2nd ed., Vol. 4, p. 93, 1960. 7. H. T. Wright, J. Kraut, and P. E. Wilcox, J M B 37, 363 (1968).
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NH,
A
6
1
(s
trypsm chyrn01ryp51nc
'I46 c COOH Neo -chymotrypinogen
NHz;~
e
A
a - Chymotrypsin
A NH-13 Chymotrypsinogen
CCQH
16
CGQH
B
y - Chymotrypsin
COOH
FIQ.1. Genesis of the various forms of chymotrypsin.
trypsin is the predominant form obtained when chymotrypsinogen is activated rapidly by relatively large amounts of trypsin. (3) Both a- and y-chymotrypsin are missing two dipeptides, Ser 14Arg 15 and Thr 147-Asn 148; a-chymotrypsin is the predominant form obtained when chymotrypsinogen is activated slowly by relatively small amounts of trypsin, and y-chymotrypsin is obtained from the slowly activated preparation only after standing for long times a t high pH, e.g., 5 days a t pH 5.6 (8). (4) Conversion of y- to a-chymotrypsin is possible but very slow, requiring several months at p H 4.0 (8). ( 5 ) Neo-chymotrypsinogen designates any one of seven theoretically possible members of the class of enzymically inactive zymogen molecules that have had one or more of the following three peptide bonds broken by chymotrypsin itself: Leu 1 3 S e r 14, Tyr 146-Thr 147 or Asn 148-Ala 149 (9). Three of the seven have been characterized, and are activatable by tryptic cleavage of the critical bond Arg 15-Ile 16. Extensive physicochemical studies have been carried out in various laboratories during the past 15 years in s n effort to learn something about the structural basis of the zymogen to enzyme conversion. Neu8. R. B. Corey, 0. Battfay, D. A. Brueckner, and F. G. Mark, BBA 94,535 (1965). 9. M. Rovery, M. Poilroux, A. Yoshida, and P. Desnuelle, BBA 23, 608 (1957).
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rath et al. (10)first observed that a change in optical rotation accompanies the appearance of enzymic activity during activation. Several more detailed investigations, utilizing ORD (11, 19) and CD (13) have resulted in a variety of interpretations as to whether or not additional helix is being formed. Hess and his collaborators have shown that the new free terminal amino group a t Ile 16 is required to maintain the enzyme in an active conformation (14), and a beautiful explanation of this observation has been provided by the X-ray structure of a-chymotrypsin, as described in Chapter 6 by Blow, with the discovery that the free -ma+ group of Ile 16 forms an internal ion pair with the side chain carboxylate group of Asp 194. 111. X-Ray Crystallographic Results
It should be emphasized at the outset that the model of chymotrypsinogen is still, a t the time this is written, in a relatively unrefined state compared to the a-chymotrypsin model. Anomalous dispersion data have been measured but have not yet been included in the refinement calculations, nor has the model been matched against the electron density map with the precision made possible by development of the Richards optical comparitor (15).For these reasons, a table of coordinates is not included in this chapter. Nevertheless, the zymogen structure is now sufficiently detailed and reliable that many interesting observations can be made concerning the stereochemistry of the activation process, and perhaps a few meaningful questions can be asked. An important point for the purposes of the chapter is that the threedimensional structures of all forms of the active enzyme (that is, the a, y, 8, and x forms) are essentially identical, a t least in the crystals and to within the resolution limits of the current X-ray diffraction data. Therefore, it is assumed throughout this chapter that all significant features of the conformational changes undergone by the zymogen when it is activated will be visible upon comparison of the structures of achymotrypsin and the zymogen, again with the usual cautionary note about resolution. (Of course, there is no guarantee implied that, because 10. H.Neurath, J. A. Rupley, and W. J. Dreyer, ABB 65, 243 (1966). 11. D.N. Raval and J. A. Schellman, BBA 107, 463 (1965). 12. R. Biltonen, R. Lumry, V. Madison, and H. Parker, Proc. Natl. Acad. Sci. U.S. 54, 1412 (1965). 13. G.Fasman, R. J. Foster, and S. Beychok, JMB 19, 240 (1968). 14. H. L. Oppenheimer, B. Labouesse, and G . P. Heas, JBC 241, 2720 (1966). 15. F. M.Richards, JMB 37, 225 (1968).
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J . KRAUT
they are visible, such significant conformational changes will necessarily have been noticed by the author.) Low resolution X-ray diffraction studies have shown that PMS-T-, PMS-8-, PMS-7-, and active y-chymotrypsin are crystallographically isomorphous and that all of the prominent features of difference Fourier maps among the various members of this group correspond to the expected missing dipeptides or added PMS inhibitor group (7, 16). Therefore, it can safely be assumed that any conformational differences between members of the T , 8 , and y group are quite minor. It is much more difficult tq decide whether there is any conformational difference between a- and 7-chymotrypsin, or whether they are merely different crystalline forms of the same molecule, since the only wellestablished distinction between a- and y-chymotrypsin is in their crystal form. The first crystallizes from 0.4 saturated (NH,),SO,, pH 4.0, in the monoclinic space group P2, with 2 molecules per asymmetric unit (i.e., as the dimer), and the latter crystallize from 0.4 saturated (NH,),SO,, pH 5.6, in the tetragonal space group P4,2,2 with one molecule per asymmetric unit (8). Figure 1 implies that there is indeed some conformational difference by showing the ends curled back a t the break between residues 146 and 149 in the representation of a-chymotrypsin as well as in neo-chymotrypsinogen. The rationale for this was essentially as follows: (1) a-chymotrypsin is known to dimerize in solution at pH 4; (2) cu-chymotrypsin crystallizes as dimers, in which T r y 146 and Ala 149 play a role in dimer formation; (3) 8-chymotrypsin has been reported not to dimerize under the same conditions; (4) 8-chymotrypsin crystallizes in a form which does not contain a twofold axis corresponding to the local dimer axis found in a-chymotrypsin crystals; ( 5 ) as already mentioned above, crystals of PMS-8-, PMS-y- and active ychymotrypsin are isomorphous. All this suggests that there is some small conformational difference between a- and y-chymotrypsin that expresses itself in their differing dimerization behavior. Nevertheless, X-ray crystallographic study (7, 17, 18) has thus far failed to reveal any obvious difference between the geometries of the two molecules. I n order to determine that a- and y-chymotrypsin really are different conformational states of the molecule rather than merely different crystal forms, it would be necessary to show th
U.8. 58, 304 (1967).
17. B. W. Matthews, G. H. Coden, E. W. Silverton, H. Braxton, and D. R. Davies, J M B 36, 179 (1968). 18. G. H. Cohen, E. W. Silverton, B. W. Matthews, H. Braxton, and D. R. Davies, J M B 44,129 (1969).
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171
e.g., that a-chymotrypsin dimerizes under conditions where 7-chymotrypsin does not or, better still, that they displayed different enzymekinetic behavior. To reiterate the point made a t the beginning of this section, then, it appears to be well established that all forms of the active enzyme have very similar if not identical conformations. This is important because comparison between the zymogen and enzyme structures has been made using the Cambridge model of a-chymotrypsin, which is essentially an autodegraded form of the initial zymogen activation product, 7-chymotrypsin. If it had happened that a-chymotrypsin assumed a different conformation from a-chymotrypsin, then we would not know which of the changes we see between the zymogen and the a molecule were due to zymogen activation, and hence presumably significant for the genesis of enzymic activity, and which were merely incidental consequences of the removal of the dipeptides Ser 14-Arg 15 and Thr 147-Asn 148 in going from 7 to a. In particular, one of the changes we believe may be significant, that at Arg 145, falls in a chain segment that includes the second of these dipeptides. We are fortunate that this question does not arise. The first land most obvious result to come out of the comparison of the zymogen and enzyme structures is that the overall folding of the two molecules is very similar. No extensive rearrangement of the backbone chain has resulted from the cleavage of the critical Arg 15-Ile 16 peptide bond. In this respect, the conclusions drawn from earlier S A crystallographic studies of the zymogen (19,,% andI the ) T-,8-, and ychymotrypsin group ($1) are fully substantiated. Thus, the general description of the conformation of a-chymotrypsin given in the chapter by Blow is also applicable to the zymogen. Both molecules are composed almost entirely of a more or less fully extended polypeptide chain which often folds back on itself to form large sections of distorted antiparallel pleated sheet. Before turning to a discussion of the details of the comparison between zymogen and enzyme structures, it would be appropriate first to dispose of one point concerning the structure of the zymogen itself. Confirming earlier conclusions based on low-resolution X-ray studies of chymotrypsinogen and the a, 8, and y family of chymotrypsins ( H ) ,the two dipeptides Ser l P A r g 15 and Thr 147-Asn 148 which are split out on con19. J. Kraut, L. s. Sieker, D. F. High, and s. T. Freer, PTOC.Natl. Acad. Sci. U.s. 48, 1417 (1962). 20. J. Kraut, D. F. High, and L. C. Sieker, Pmc. Natl. Acad. Sci. U.S. 51, 839 (1964). 21. J. Kraut, H. T. Wright, M. Kellerman, and U.S. 58, 304 (1967).
S. T. Freer, Proc. Natl. Acud. J%i.
172
J. KRAUT
version to 8- and a- or y-chymotrypsin are located on the surface of the zymogen molecule and are easily accessible to enzymic attack. Indeed, the side chain of Arg 15 is pointing straight out into the surrounding medium and is an excellent candidate for attack by the activating enzyme, trypsin. Interestingly, chymotrypsinogen contains three other arginine residues, 145, 154, and 230, but the side chains of all three lie in surface crevices in our present model. However, there are in addition 14 lysine residues in chymotrypsinogen, and the side chains of most of these project outward. There is no obvious reason why some of them should not be rapidly attacked by trypsin as well. We return now to compare the zymogen and active enzyme in detail. The high degree of similarity between the two molecules is readily apparent by comparison of Figs. 2 and 3, drawn from computer-plotted
FIQ.2. Chymotrypsinogen-simplified backbone chain linking the a-carbon atoms of each amino acid residue, drawn from a vantage point looking toward the latent catalytic site region. The side chains of some important amino acid residues are also shown. The drawing was prepared from a computer-plotted perspective projection of all a-carbon atoms.
5.
CHYMOTRYPSINOGEN : X-RAY STBUCTURE
173
FIQ.3. a-Chymotrypsin-simplified backbone chain linking the a-carbon atoms of each amino acid residue, drawn from a vantage point equivalent to that of Fig. 2. The side chains of some important amino acid residues are also shown. This drawing and all subsequent a-chymotrypsin drawings were prepared from the coordinates of tosyl-cu-chymotrypsin. Accordingly, the side chain of Ser 196 ia shown in a position appropriate to the inhibited enzyme. In native a-chymotrypsin this side chain is rotated slightly about its a-/3 bond and aeaumes a position close to that shown for the zymogen in Fig. 2. The drawing waa prepared from a computer-plotted perspective projection of the a-carbon atoms. A portion of the chain from residues 9-13 is not shown because of the uncertainty in position of these residues (,%I.
perspective projections of the a-carbon coordinates for the zymogen (Fig. 2) and for tosyl-a-chymotrypsin (22) (Fig. 3). The coordinates of the latter were first rotated to minimize the sum of squares of distances between corresponding a-carbon atoms in the two molecules. The mean displacement between all corresponding a-carbon atoms is only 22. J. J. Birktoft, B. W. Matthews, and D. M. Blow,
BBRC 36, 131
(1!369).
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J. KRAUT
1.8 A, which may be considered as some sort of index of how slight the difference in overall conformation actually is. There are, however, a few segments of backbone chain that move considerably upon activation. Table I lists all residues for which the a-carbon atom is displaced by more than 3.6A, a cutoff chosen rather arbitrarily because the table can then be readily divided into groups of contiguous residues and shows which chain segments have moved the most. Some of these movements have no apparent relationship to the activation process. Thus, segments I, 111, and IV are exterior loops of backbone chain, far from the active site, in both zymogen and enzyme. Indeed, segment IV, where one of the largest movements occurs, is probably a flexible region since it is known to assume different conformations in the two molecules of the a-chymotrypsin asymmetric unit (22). Therefore, this particular conformational difference between the zymogen and enzyme may be simply a reflection of differing crystal packing forces and
TABLE I RESIDUESFOR WHICH a-CARBONATOMSDIFFERIN POSITION BY Mom THAN 3.6 A BETWEEN THE CHYMOTRYPSINOGEN AND LY-CHYMOTRYPSIN STRUCTURES Segment
Displacement (A)
I
Gln 7 Pro 8
4.8 10.0"
11
Ile 16 Val 17
11.3 6.6
I11
Thr 37 Gly 38
4.0 6.6
IV
h p 72 Gln 73 Gly 74 Ser 75 Ser 76 Ser 77
5.6 9.6 9.1 6.2 10.1 5.6
V
Thr 144 Arg 145 Tyr 146 Ala 149 h n 150 Thr 151 Pro 152
5.9 8.7 4.6 4.7 6.7' 4.74 4.6"
Met 192 Gly 193
8.4 6.6
VI a
Residue
Tentative chymotrypsinogen coordinates.
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CHYMOTRYPSINOGEN: X-RAY STRUCTURE
175
in actuality have nothing to do with the activation process. The same is possibly true for segments I and I11 as well. A conformational change not included in Table I since the greatest a-carbon movement is only 3.4A a t residue 173 may be mentioned in passing. It involves residues 161-173. I n the enzyme, this chain segment forms two turns of distorted but nevertheless unquestionable helix, which was overlooked initially but subsequently pointed out by Blow (23). I n the zymogen this helical segment is further distorted so that it probably cannot be said to form more than one turn of helix and to include more than residues 164-168. This is the only part of the zymogen molecule where an alteration of helix content may occur upon activation. We now turn our attention to segments 11,V, and VI in Table I. These are almost certainly significant in the sense that they are required for conversion of the inactive zymogen into an active enzyme. A. ISOLEXJCINE 16 The conformational change in segment I1 brings the newly formed N-terminus of Ile 16 into the interior of the molecule where it approaches the buried side chain carboxylate group of Asp 194. This movement can be seen by comparing Figs. 2 and 3 and in greater detail by comparing Figs. 4 and 5. I n the latter pair of figures, note that residues 16 and 17 are above the chain segment 18-21 in the zymogen but below segment 18-21 in the enzyme. Further, in the zymogen, Asp 194 is buried to begin with, and its side chain carboxylate forms a hydrogen bond with Nc2 of His 40. I n the enzyme, on the other hand, the Asp 194 side chain has swung around by about 4 A into a position where it can hydrogen bond with the new Ile 16 N-terminus. Note also that the imidazole ring of His 40 has simultaneously turned slightly and now donates a hydrogen bond from Ne2 to the carbonyl oxygen of Gly 193. Apparently, the large movement of segment I1 is accomplished by rotating the m.ain chain approximately 180" about an axis roughly defined by the a-carbon atoms of residues 19 and 21. This rotation swings the new N-terminus of the B chain out through the surrounding solvent, and then down into the interior of the molecule, burying the formerly exposed side chains of Ile 16 and Val 17 just below the surface of the enzyme molecule. The finding that Asp 194 is buried in the zymogen as well as in the enzyme is in agreement with the chemical modification studies of Ciarraway et al. (244). 23. D. M.Blow, Biochem. J . 112,261 (1969). 24. K.L. Carraway, P. Spoerl, and D. E.Koshland, J M B 42, 133 (1969).
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J. KRAUT
F I ~4.. Chymotrypsinogen-stereoscopic projection of amino acid residues involved in the formation of the Ile 1bAsp 194 ion pair during activation. The figure is drawn from a vantage point above the surface looking toward the interior of the molecule (from top to bottom on Fig. 2). Figures 4 through 9 were prepared from computer-plotted stereoscopic projections.
B. ARGININE145 The most obvious result of the conformational change in segment V is to move the guanidinium side chain of Arg 145 away from the neighborhood of His 40,Asp 194,and the catalytic site Ser 195 and out into the surrounding medium. This movement is clearly evident upon comparison of Figs. 6 and 7. I n the zymogen, the guanidinium group is resting on the surface of the molecule and may be close enough to the buried side chain carboxylate of Asp 194 to permit appreciable electrostatic interaction between these two oppositely charged groups. Possibly this electrostatic interaction helps to stabilize the structure. I n the enzyme, on the other hand, segment V is rearranged so that the a-carbon of Arg 145 has moved by 9A, and its side chain has swung up to become fully extended into the solvent. That a positively charged side chain in this location may play
5.
CHYMOTRYPSINOGEN : X-RAY STRUCTURE
177
0 -
FIG.5. a-Chymotrypsin-stereoscopic projection of amino acid residues involved in the formation of the Ile 16-Asp 194 ion pair during activation. The figure ia drawn from a vantage point equivalent to that of Fig. 4.
FIQ.6. Chymotrypsinogen-stereoscopic projection of the latent catalytic site region drawn from a vantage point looking into the page (see Fig. 2) at an angle inclined about 20" to the right of perpendicular.
178
J. KRAUT
0 0
cmm OXYGEN NITROGEN
0
p
ASP 102
SULFUR
ASP 102
FIQ.7. a-Chymotrypsin-stereoscopic projection of the catalytic site region drawn from a vantage point equivalent to that of Fig. 6.
some role in the activation process is consistent with the fact that the corresponding residue is also an arginine in pig elastase and bovine thrombin or a lysine in bovine trypsinogen and chymotrypsinogen B (26,26).It must be cautioned before drawing any final conclusions, however, that the electron density map in the region of the Arg 145 side chain is somewhat ambiguous for the zymogen and that an alternative model can be constructed with the side chain extending outward. However, the electron density in the alternative position is much weaker. Perhaps this indicates some degree of disorder with the side chain actually occupying two different positions. In any case, it is difficult to imagine 25. M. 0. Dayhoff, ed., “Atlas of Protein Sequence and Structure” Vol. 4, p. DB4. Natl. Biomed. Res. Found., Silver Spring, Maryland, 1969. 26. S. Magnusson, in “Structure-Function Relationships of Proteolytic Enzymes” (P. Desnuelle, H. Neurath, and M. Ottesen, eds.), p. 138. Munksgaard, Copenhagen, 1970.
5.
CHYMOTRYPSINOGEN : X-RAY STRUCTURE
179
a role for this conformational change in segment V given the alternative position for the Arg 145 side chain.
C. METHIONINE 192 Although segment VI in Table I includes only Met 192 and Gly 193, the conformational change represented here is of considerable interest since it is the only one to which a fairly clear-cut role in the genesis of enzymic activity can be assigned. It results in movement of the side chain of Met 192 from a deeply buried position in the zymogen out to the surface of the enzyme molecule where it forms the flexible hydrophobic lid of the specificity cavity (27). This movement is most clearly visualized by comparing Figs. 6 and 7 but may also be seen in the other figures as well. In the process of moving Met 192 out to the molecular surface, segment 187-193 of the main chain also becomes more extended. The consequence of these combined conformational changes is the creation of the specificity cavity, one side of which is comprised of residues 189192 (27). In the zymogen this cavity does not exist, or a t least it is incomplete and severely distorted. The molecular contortions involved here may be thought of in a simplified way as resulting from a rotation of the main chain about the carbonyl-carbon to a-carbon bond of Asp 194. This rotation causes the shift ,in position of the Asp 194 side chain from the vicinity of His 40 toward the buried N-terminus of Ile 16, as described above, and simultaneously it also moves the backbone chain carbonyl oxygen of Gly 193 into position to accept a hydrogen bond from Nr2 of His 40,replacing the carboxylate oxygen of Asp 194. Thus, the conformational changes involving Ile 16, Met 192, Gly 193, and Asp 194 and formation of the specificity cavity during zymogen activation can be viewed as a single unified event. An attempt has been made to depict certain aspects of the specificity cavity formation from a third vantage point in Figs. 8 and 9. I n Fig. 9, the tosyl group occupying the specificity cavity in tosyl-a-chymotrypsin is shown in black.
D. CATALYTIC SITE As described in the chapters by Blow and by Hess on chymotrypsin, a mechanism for the bond breaking step in the catalytic pathway has been proposed which employs a “charge relay system” (27,28) involving a hydrogen bond network between the side chains of Ser 195, His 57, and Asp 102. It has been very surprising therefore, to find that the spatial arrange27. T.A. Steitz, R. Henderson, and D. M. Blow, J M B 46, 337 (1969). 28. D.M.Blow, J. J. Birktoft, and B. S. Eartley, Nature a21, 337 (1969).
180
J. KRAUT
FIQ.8. Chymotrypsinogen-stereoscopic projection of the latent specificity cavity drawn from roughly the same vantage point as Fig. 2.
F’IQ.9. Tosyl-cr-chymotrypsin-stereoscopic projection of the specificity cavity drawn from a vantage point equivalent to that of Fig. 8. The tosyl group is drawn with solid bonds to contrast with the bonds of the amino acids forming the specificity cavity.
5. CHYMOTRYPSINOGEN X-RAY STRUCTURE
181
ment of these three residues in the zymogen is almost indistinguishable from that in the catalytically active enzyme. The term “almost indistinguishable” has been used here because, in fact, a very slight twist may actually occur in the His 57 side chain, and indeed such a twist may be seen by careful comparison of Figs. 2,6, and 8 with the corresponding Figs. 3,7, and 9. If this effectis real, it may suffice to explain the total inactivity of chymotrypsinogen since it would result in distortion of the hydrogen bond network referred to above. Wang (2%) has argued convincingly that facilitated proton transfer along precisely aligned hydrogen bonds may play a crucial role in enzyme catalysis generally. One must be careful, however, to emphasize that the apparent twist in the His 57 side chain is so small that it could well turn out to be spurious when the zymogen electron density map is refined or when direct comparison of the two maps is carried out. Indeed, other subtle but significant structural changes in the charge relay system could also be revealed by further improvement in the data. Nevertheless, at the present stage of the X-ray study of chymotrypsinogen, one can say with reasonable certainty that the catalytic site, in contrast with the specificity cavity, is essentially preformed. The question remains, therefore, as to why the zymogen is not catalytically active, though perhaps without the specificity for hydrophobic side chains characteristic of chymotrypsin. Further to pursue this question, it should be pointed out that two minor structural changes which may also help to explain the zymogen’s inactivity are seen upon activation. These occur not in the charge relay system itself but in its immediate neighborhood: (1) the side chain of Ile 99 moves away from the imidazole ring of His 57, and (2) the hydroxyl group of Ser 214 moves into a better position to form a second hydrogen bond with 082 of Asp 102.These will now be described in more detail. The displacement of the side chain of Ile 99 upon activation is quite noticeable and readily seen on comparing Figs. 6 and 7. I n the zymogen, the Ile 99 side chain is in van der Waals contact with the imidazole ring of His 57 and, together with neighboring side chains, completely blocks access of the solvent to His 57-Asp 102 portion of the catalytic site hydrogen bond network. During activation, the Ile 99 side chain rotates approximately 90” about its a-carbon to P-carbon bond and moves away from the His 57 ring. Curiously, the conformation of the main chain does not alter appreciably. Just why, or even whether or not, this change would be required for inducing enzymic activity is open to question. Such an alteration might be expected, if anything, to decrease the hydro29. J. H. Wang, Science 161, 328 (1968).
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phobicity of the region surrounding His 57-Asp 102, which would in fact appear to be a change in the wrong direction. It has been suggested that the hydrophobic environment of the buried Asp 102 enhances the polarization of the charge relay system and makes the reactive Ser 195 oxygen strongly nucleophilic as required by the proposed mechanism (28).Nevertheless, it is noteworthy that a hydrophobic residue (Ile, Leu, or Val) is conserved a t the position corresponding to Ile 99 in bovine chymotrypsinogen B, bovine trypsinogen, bovine thrombin, and pig elastase (26, 6 6 ) . The change a t Ser 214 is more subtle and at the present resolution remains somewhat doubtful. It involves a possible movement of Oy by perhaps 1 A to form a better hydrogen bond with 062 of Asp 102. In itself, this is probably not a sufficiently large apparent movement to be taken seriously. However, there does occur a rather obvious change in conformation of the chain segment 214-217 during activation. In the zymogen, the stretch of main chain running from the a-carbon of residue 214 to the a-carbon of residue 216 is somewhat folded, but it is fully extended in the enzyme. The effect can be observed readily by comparing Figs. 2 and 3. In light of the proposal by Steitz e t al. (27) that the carbonyl oxygen of Ser 214 is involved in productive binding of substrates, one might expect to see this carbonyl group move into binding position as a result of the main chain extension, but such is not the case. The carbonyl group is in about the same orientation in both zymogen and enzyme. On the other hand, there are several observations suggesting that a second serine residue (Ser 214 in the case of chymotrypsin) forms a hydrogen bond with the same buried aspartic side chain oxygen (082 of Asp 102) that is also hydrogen bonded to the catalytic site histidine side chain (N61 of His 57) in serine proteases generally. This point is discussed more fully in Chapter 15. If this view is correct, the second serine hydrogen bond may well be included in the catalytic site hydrogen bond network and play some role in the activity of these enzymes. It is all the more tempting, therefore, to suppose that distortion of this bond in chymotrypsinogen is one of the factors responsible for its lack of activity and provisionally to consider as significant the apparent slight movement of the Ser 214 side chain.
E. ACTIVATION RDFOLDINQ It will be obvious that at least as many questions about the chymotrypsinogen activation process have been raised as have been answered by the observations reported here. Indeed, the central problem of why the
5. CHYMOTRYPSINOGEN : X-RAY STRUCTURE
183
aymogen is inactive remains, although it seems reasonably certain that the answer will involve some subtle distortions in and around the catalytic site. Perhaps a suitable way to conclude this chapter would be to mention briefly another, and no doubt more difficult, problem that now arises. Why does the simple act of cleaving a single exterior peptide bond between Arg 15 and Ile 16 cause the chymotrypsinogen molecule to refold in the manner observed? The answer is certainly not obvious from inspection of the two models, and we shall have to content ourselves with simply listing those structural changes which probably contribute, either positively or negatively, to the overall net free-energy decrease upon going from the aymogen to the enzyme conformation. (1) The Arg 15-Ile 16 peptide bond is hydrolyzed to give a free -COOand a free -NH,+ group. (2) The free -NH,+ group tucks down inside the molecule where it interacts with the buried -COO- group of Asp 194. (3) As a result of this process two hydrophobic side chains, a t residues 16 and 17, become buried as well. (4) The buried -COO- group of Asp 194 moves away from the His 40 side chain to which it had been hydrogen bonded and is replaced by the backbone carbonyl of Gly 193 which forms a new hydrogen bond with the His 40 side chain. ( 5 ) The positively charged side chain of Arg 145 moves away from the surface of the molecule, where it may interact with the buried COOof Asp 194, and extends out into the surrounding solvent. (6) The buried hydrophobic side chain of Met 192 moves from the molecular interior out to the surface.
Other conformational changes also occur, some of them quite large, as, for example, in segments I, 111, and I V of Table I. But unlike the events enumerated above for which the situation is fairly clear, it has not yet been possible to see how they might contribute to the free energy of refolding.
.
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
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. . . .
165 167 169 175 176 179 179 182
1. Introduction
The material to be covered here would logically have been incorporated into the chapter by Blow on chymotrypsin, instead of being presented as a relatively independent entity, except for the circumstance that the results described herein were still being prepared for publication while that chapter was being written. Therefore, sacrificing organizational elegance in the interest of currency, the authors of the two chapters have chosen to describe the three-dimensional structure of bovine chymotrypsinogen A and its relationship to the activation process separately. Throughout the rest of this chapter, bovine chymotrypsinogen A will be referred to simply as chymotrypsinogen or the zymogen. Almost all the material discussed here is based upon the paper by Freer et al. (I), wherein the preliminary results of the X-ray analysis of chymotrypsinogen at 2.5A resolution are presented. It is important to 1. S. T.Freer, J. Kraut, J. D. Robertus, H. T. Wright, and Ng. H. Xuong, chemistry 9, 1997 (1970). 166
BW-
166
J. KRAUT
emphasize the word preliminary since experience has shown that protein structures determined by X-ray analysis have a decided tendency to undergo continuous minor modification over a period of years as small errors in the original model are discovered, electron density maps are gradually improved, errors in amino acid sequences are corrected, water molecules are discovered, and, most especially, as the investigators come to see subtle but significant features that may have escaped notice in the first burst of enthusiasm over a newly determined structure. I n the case of chymotrypsinogen, even as this is being written more careful comparison of the enzyme and zymogen structure is being carried out both in Cambridge and La Jolla, and improved electron density maps are being computed; thus, there is little doubt that the story presented here will soon prove to be incomplete. Nevertheless, some interesting and thought-provoking observations have already been made and will be summarized in this chapter. The phenomenon of induction of biological activity by limited proteolysis of an inactive precursor is fairly widespread in nature. It has been found in various forms in such widely separated types of organisms as bacteria, yeast, green plants, invertebrates, and vertebrates. The subject has been reviewed recently in detail by Ottesen (2), and so it need not be extensively surveyed here. The point to be made is that in a sense it represents a kind of biochemical amplification and control mechanism of which the conversion of chymotrypsinogen to chymotrypsin in the vertebrate duodenum, under the influence of the enzyme trypsin, is only one relatively simpIe though well-known example. Another much more complex example of limited proteolysis as a biological amplification and control mechanism is seen in the clotting blood. Here, a t least eight distinct factors interact in a cascading sequence that ultimately results in the conversion of soluble fibrinogen into an insoluble stabilized fibrin clot (3).A perhaps even more complex case familiar to the immunologist is that of the complement system. Enzymic cleavage is an essential feature of the activation mechanism of several complement components, of which 11 are now recognized ( 4 ) . Nor is the phenomenon of activation by limited proteolysis restricted to enzymes. The hormone insulin, which plays an important role in glucose metabolism, is now known to be the product of limited proteolysis of a single-chain proinsulin molecule. The latter is synthesized in specialized cells of the pancreas, where it is activated prior to secretion by the splitting out of a polypeptide connecting 2. M. Ottesen, Ann. Rev. Biochem. 36, 55 (1967). 3. E. W. Davie and 0. D. Ratnoff, in “The Proteins” (H. Neurath, ed.), 2nd ed., Vol. 3, p. 359. Academic Press, New York, 1965. 4. H . J. Muller-Eberhard, Ann. Rev. Biochem. 38, 389 (1969).
5.
CHYMOTRYPSINOGEN : X-RAY STRUCTURE
167
the C-terminus of the insulin B chain with the N-terminus of the A chain (6). Thus, with the detailed architecture of the chymotrysinogen molecule now becoming clear, and the model of chymotrypsin itself already well established, we have our first glimpse of one simple example of this important type of biological phenomenon a t the level of molecular structure, II. The Activation Process
There are several steps involved in the process of activating chymotrypsinogen to give the various recognized forms of chymotrypsin. It is worthwhile considering what is known of this process and how the various chymotrypsins are related in order to help evaluate the significance of the structural differences, to be described below, between the zymogen and the a form of the active enzyme. A review of the chemistry of the activation process as it was understood in 1960 has been given by Desnuelle (6) in the previous edition of “The Enzymes.” Although Desnuelle’s review antedated the complete sequence determination, from the point of view of classical enzyme chemistry there is not very much that is new to be said about the subject; thus it will be only briefly summarized here. Figure 1 is reproduced from Wright et a2. (7).It is a suggested modification of the scheme generally accepted until now (6) but will nevertheless serve as a convenient summary of the nomenclature and chemical relationships among the forms of chymotrypsin. The proposed scheme differs from the classical one in that it supposes a-chymotrypsin to be conformationally different from 7-chymotrypin, and to be produced only by activation of the neo-chymotrypsinogen that has the dipeptide Thr 147-Asn 148 deleted, and not directly by degradation of S-chymotrypsin. Well-established features of the activation scheme are as follows: (1) Chymotrypsinogen is converted to the fully active n-chymotrypsin by trypsin catalyzed hydrolysis of the peptide bond between Arg 15 and Ile 16. It is this peptide-bond scission, and this one alone, that generates enzymic activity. All subsequent products result from autodegradation of the n-chymotrypsin molecule. (2) S-Chymotrypsin is missing the dipeptide Ser 14-Arg 15; S-chymo5. D. F. Steiner, J. L. Clark, C. Nolan, A. R. Rubenstein, E. Margobash, B. Aten, and P. E. Oyer, Recent Progr. Hormone Rea. 25, 207 (1969). 6. P. Desnuelle, “The Enzymes,” 2nd ed., Vol. 4, p. 93, 1960. 7. H. T. Wright, J. Kraut, and P. E. Wilcox, J M B 37, 363 (1968).
168
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NH,
A
6
1
(s
trypsm chyrn01ryp51nc
'I46 c COOH Neo -chymotrypinogen
NHz;~
e
A
a - Chymotrypsin
A NH-13 Chymotrypsinogen
CCQH
16
CGQH
B
y - Chymotrypsin
COOH
FIQ.1. Genesis of the various forms of chymotrypsin.
trypsin is the predominant form obtained when chymotrypsinogen is activated rapidly by relatively large amounts of trypsin. (3) Both a- and y-chymotrypsin are missing two dipeptides, Ser 14Arg 15 and Thr 147-Asn 148; a-chymotrypsin is the predominant form obtained when chymotrypsinogen is activated slowly by relatively small amounts of trypsin, and y-chymotrypsin is obtained from the slowly activated preparation only after standing for long times a t high pH, e.g., 5 days a t pH 5.6 (8). (4) Conversion of y- to a-chymotrypsin is possible but very slow, requiring several months at p H 4.0 (8). ( 5 ) Neo-chymotrypsinogen designates any one of seven theoretically possible members of the class of enzymically inactive zymogen molecules that have had one or more of the following three peptide bonds broken by chymotrypsin itself: Leu 1 3 S e r 14, Tyr 146-Thr 147 or Asn 148-Ala 149 (9). Three of the seven have been characterized, and are activatable by tryptic cleavage of the critical bond Arg 15-Ile 16. Extensive physicochemical studies have been carried out in various laboratories during the past 15 years in s n effort to learn something about the structural basis of the zymogen to enzyme conversion. Neu8. R. B. Corey, 0. Battfay, D. A. Brueckner, and F. G. Mark, BBA 94,535 (1965). 9. M. Rovery, M. Poilroux, A. Yoshida, and P. Desnuelle, BBA 23, 608 (1957).
5.
CHYMOTRYPSINOGEN: X-RAY STRUCTURE
169
rath et al. (10)first observed that a change in optical rotation accompanies the appearance of enzymic activity during activation. Several more detailed investigations, utilizing ORD (11, 19) and CD (13) have resulted in a variety of interpretations as to whether or not additional helix is being formed. Hess and his collaborators have shown that the new free terminal amino group a t Ile 16 is required to maintain the enzyme in an active conformation (14), and a beautiful explanation of this observation has been provided by the X-ray structure of a-chymotrypsin, as described in Chapter 6 by Blow, with the discovery that the free -ma+ group of Ile 16 forms an internal ion pair with the side chain carboxylate group of Asp 194. 111. X-Ray Crystallographic Results
It should be emphasized at the outset that the model of chymotrypsinogen is still, a t the time this is written, in a relatively unrefined state compared to the a-chymotrypsin model. Anomalous dispersion data have been measured but have not yet been included in the refinement calculations, nor has the model been matched against the electron density map with the precision made possible by development of the Richards optical comparitor (15).For these reasons, a table of coordinates is not included in this chapter. Nevertheless, the zymogen structure is now sufficiently detailed and reliable that many interesting observations can be made concerning the stereochemistry of the activation process, and perhaps a few meaningful questions can be asked. An important point for the purposes of the chapter is that the threedimensional structures of all forms of the active enzyme (that is, the a, y, 8, and x forms) are essentially identical, a t least in the crystals and to within the resolution limits of the current X-ray diffraction data. Therefore, it is assumed throughout this chapter that all significant features of the conformational changes undergone by the zymogen when it is activated will be visible upon comparison of the structures of achymotrypsin and the zymogen, again with the usual cautionary note about resolution. (Of course, there is no guarantee implied that, because 10. H.Neurath, J. A. Rupley, and W. J. Dreyer, ABB 65, 243 (1966). 11. D.N. Raval and J. A. Schellman, BBA 107, 463 (1965). 12. R. Biltonen, R. Lumry, V. Madison, and H. Parker, Proc. Natl. Acad. Sci. U.S. 54, 1412 (1965). 13. G.Fasman, R. J. Foster, and S. Beychok, JMB 19, 240 (1968). 14. H. L. Oppenheimer, B. Labouesse, and G . P. Heas, JBC 241, 2720 (1966). 15. F. M.Richards, JMB 37, 225 (1968).
170
J . KRAUT
they are visible, such significant conformational changes will necessarily have been noticed by the author.) Low resolution X-ray diffraction studies have shown that PMS-T-, PMS-8-, PMS-7-, and active y-chymotrypsin are crystallographically isomorphous and that all of the prominent features of difference Fourier maps among the various members of this group correspond to the expected missing dipeptides or added PMS inhibitor group (7, 16). Therefore, it can safely be assumed that any conformational differences between members of the T , 8 , and y group are quite minor. It is much more difficult tq decide whether there is any conformational difference between a- and 7-chymotrypsin, or whether they are merely different crystalline forms of the same molecule, since the only wellestablished distinction between a- and y-chymotrypsin is in their crystal form. The first crystallizes from 0.4 saturated (NH,),SO,, pH 4.0, in the monoclinic space group P2, with 2 molecules per asymmetric unit (i.e., as the dimer), and the latter crystallize from 0.4 saturated (NH,),SO,, pH 5.6, in the tetragonal space group P4,2,2 with one molecule per asymmetric unit (8). Figure 1 implies that there is indeed some conformational difference by showing the ends curled back a t the break between residues 146 and 149 in the representation of a-chymotrypsin as well as in neo-chymotrypsinogen. The rationale for this was essentially as follows: (1) a-chymotrypsin is known to dimerize in solution at pH 4; (2) cu-chymotrypsin crystallizes as dimers, in which T r y 146 and Ala 149 play a role in dimer formation; (3) 8-chymotrypsin has been reported not to dimerize under the same conditions; (4) 8-chymotrypsin crystallizes in a form which does not contain a twofold axis corresponding to the local dimer axis found in a-chymotrypsin crystals; ( 5 ) as already mentioned above, crystals of PMS-8-, PMS-y- and active ychymotrypsin are isomorphous. All this suggests that there is some small conformational difference between a- and y-chymotrypsin that expresses itself in their differing dimerization behavior. Nevertheless, X-ray crystallographic study (7, 17, 18) has thus far failed to reveal any obvious difference between the geometries of the two molecules. I n order to determine that a- and y-chymotrypsin really are different conformational states of the molecule rather than merely different crystal forms, it would be necessary to show th
U.8. 58, 304 (1967).
17. B. W. Matthews, G. H. Coden, E. W. Silverton, H. Braxton, and D. R. Davies, J M B 36, 179 (1968). 18. G. H. Cohen, E. W. Silverton, B. W. Matthews, H. Braxton, and D. R. Davies, J M B 44,129 (1969).
5.
CHYMOTRYPSINOGEN : X-RAY STRUCTURE
171
e.g., that a-chymotrypsin dimerizes under conditions where 7-chymotrypsin does not or, better still, that they displayed different enzymekinetic behavior. To reiterate the point made a t the beginning of this section, then, it appears to be well established that all forms of the active enzyme have very similar if not identical conformations. This is important because comparison between the zymogen and enzyme structures has been made using the Cambridge model of a-chymotrypsin, which is essentially an autodegraded form of the initial zymogen activation product, 7-chymotrypsin. If it had happened that a-chymotrypsin assumed a different conformation from a-chymotrypsin, then we would not know which of the changes we see between the zymogen and the a molecule were due to zymogen activation, and hence presumably significant for the genesis of enzymic activity, and which were merely incidental consequences of the removal of the dipeptides Ser 14-Arg 15 and Thr 147-Asn 148 in going from 7 to a. In particular, one of the changes we believe may be significant, that at Arg 145, falls in a chain segment that includes the second of these dipeptides. We are fortunate that this question does not arise. The first land most obvious result to come out of the comparison of the zymogen and enzyme structures is that the overall folding of the two molecules is very similar. No extensive rearrangement of the backbone chain has resulted from the cleavage of the critical Arg 15-Ile 16 peptide bond. In this respect, the conclusions drawn from earlier S A crystallographic studies of the zymogen (19,,% andI the ) T-,8-, and ychymotrypsin group ($1) are fully substantiated. Thus, the general description of the conformation of a-chymotrypsin given in the chapter by Blow is also applicable to the zymogen. Both molecules are composed almost entirely of a more or less fully extended polypeptide chain which often folds back on itself to form large sections of distorted antiparallel pleated sheet. Before turning to a discussion of the details of the comparison between zymogen and enzyme structures, it would be appropriate first to dispose of one point concerning the structure of the zymogen itself. Confirming earlier conclusions based on low-resolution X-ray studies of chymotrypsinogen and the a, 8, and y family of chymotrypsins ( H ) ,the two dipeptides Ser l P A r g 15 and Thr 147-Asn 148 which are split out on con19. J. Kraut, L. s. Sieker, D. F. High, and s. T. Freer, PTOC.Natl. Acad. Sci. U.s. 48, 1417 (1962). 20. J. Kraut, D. F. High, and L. C. Sieker, Pmc. Natl. Acad. Sci. U.S. 51, 839 (1964). 21. J. Kraut, H. T. Wright, M. Kellerman, and U.S. 58, 304 (1967).
S. T. Freer, Proc. Natl. Acud. J%i.
172
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version to 8- and a- or y-chymotrypsin are located on the surface of the zymogen molecule and are easily accessible to enzymic attack. Indeed, the side chain of Arg 15 is pointing straight out into the surrounding medium and is an excellent candidate for attack by the activating enzyme, trypsin. Interestingly, chymotrypsinogen contains three other arginine residues, 145, 154, and 230, but the side chains of all three lie in surface crevices in our present model. However, there are in addition 14 lysine residues in chymotrypsinogen, and the side chains of most of these project outward. There is no obvious reason why some of them should not be rapidly attacked by trypsin as well. We return now to compare the zymogen and active enzyme in detail. The high degree of similarity between the two molecules is readily apparent by comparison of Figs. 2 and 3, drawn from computer-plotted
FIQ.2. Chymotrypsinogen-simplified backbone chain linking the a-carbon atoms of each amino acid residue, drawn from a vantage point looking toward the latent catalytic site region. The side chains of some important amino acid residues are also shown. The drawing was prepared from a computer-plotted perspective projection of all a-carbon atoms.
5.
CHYMOTRYPSINOGEN : X-RAY STBUCTURE
173
FIQ.3. a-Chymotrypsin-simplified backbone chain linking the a-carbon atoms of each amino acid residue, drawn from a vantage point equivalent to that of Fig. 2. The side chains of some important amino acid residues are also shown. This drawing and all subsequent a-chymotrypsin drawings were prepared from the coordinates of tosyl-cu-chymotrypsin. Accordingly, the side chain of Ser 196 ia shown in a position appropriate to the inhibited enzyme. In native a-chymotrypsin this side chain is rotated slightly about its a-/3 bond and aeaumes a position close to that shown for the zymogen in Fig. 2. The drawing waa prepared from a computer-plotted perspective projection of the a-carbon atoms. A portion of the chain from residues 9-13 is not shown because of the uncertainty in position of these residues (,%I.
perspective projections of the a-carbon coordinates for the zymogen (Fig. 2) and for tosyl-a-chymotrypsin (22) (Fig. 3). The coordinates of the latter were first rotated to minimize the sum of squares of distances between corresponding a-carbon atoms in the two molecules. The mean displacement between all corresponding a-carbon atoms is only 22. J. J. Birktoft, B. W. Matthews, and D. M. Blow,
BBRC 36, 131
(1!369).
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J. KRAUT
1.8 A, which may be considered as some sort of index of how slight the difference in overall conformation actually is. There are, however, a few segments of backbone chain that move considerably upon activation. Table I lists all residues for which the a-carbon atom is displaced by more than 3.6A, a cutoff chosen rather arbitrarily because the table can then be readily divided into groups of contiguous residues and shows which chain segments have moved the most. Some of these movements have no apparent relationship to the activation process. Thus, segments I, 111, and IV are exterior loops of backbone chain, far from the active site, in both zymogen and enzyme. Indeed, segment IV, where one of the largest movements occurs, is probably a flexible region since it is known to assume different conformations in the two molecules of the a-chymotrypsin asymmetric unit (22). Therefore, this particular conformational difference between the zymogen and enzyme may be simply a reflection of differing crystal packing forces and
TABLE I RESIDUESFOR WHICH a-CARBONATOMSDIFFERIN POSITION BY Mom THAN 3.6 A BETWEEN THE CHYMOTRYPSINOGEN AND LY-CHYMOTRYPSIN STRUCTURES Segment
Displacement (A)
I
Gln 7 Pro 8
4.8 10.0"
11
Ile 16 Val 17
11.3 6.6
I11
Thr 37 Gly 38
4.0 6.6
IV
h p 72 Gln 73 Gly 74 Ser 75 Ser 76 Ser 77
5.6 9.6 9.1 6.2 10.1 5.6
V
Thr 144 Arg 145 Tyr 146 Ala 149 h n 150 Thr 151 Pro 152
5.9 8.7 4.6 4.7 6.7' 4.74 4.6"
Met 192 Gly 193
8.4 6.6
VI a
Residue
Tentative chymotrypsinogen coordinates.
5.
CHYMOTRYPSINOGEN: X-RAY STRUCTURE
175
in actuality have nothing to do with the activation process. The same is possibly true for segments I and I11 as well. A conformational change not included in Table I since the greatest a-carbon movement is only 3.4A a t residue 173 may be mentioned in passing. It involves residues 161-173. I n the enzyme, this chain segment forms two turns of distorted but nevertheless unquestionable helix, which was overlooked initially but subsequently pointed out by Blow (23). I n the zymogen this helical segment is further distorted so that it probably cannot be said to form more than one turn of helix and to include more than residues 164-168. This is the only part of the zymogen molecule where an alteration of helix content may occur upon activation. We now turn our attention to segments 11,V, and VI in Table I. These are almost certainly significant in the sense that they are required for conversion of the inactive zymogen into an active enzyme. A. ISOLEXJCINE 16 The conformational change in segment I1 brings the newly formed N-terminus of Ile 16 into the interior of the molecule where it approaches the buried side chain carboxylate group of Asp 194. This movement can be seen by comparing Figs. 2 and 3 and in greater detail by comparing Figs. 4 and 5. I n the latter pair of figures, note that residues 16 and 17 are above the chain segment 18-21 in the zymogen but below segment 18-21 in the enzyme. Further, in the zymogen, Asp 194 is buried to begin with, and its side chain carboxylate forms a hydrogen bond with Nc2 of His 40. I n the enzyme, on the other hand, the Asp 194 side chain has swung around by about 4 A into a position where it can hydrogen bond with the new Ile 16 N-terminus. Note also that the imidazole ring of His 40 has simultaneously turned slightly and now donates a hydrogen bond from Ne2 to the carbonyl oxygen of Gly 193. Apparently, the large movement of segment I1 is accomplished by rotating the m.ain chain approximately 180" about an axis roughly defined by the a-carbon atoms of residues 19 and 21. This rotation swings the new N-terminus of the B chain out through the surrounding solvent, and then down into the interior of the molecule, burying the formerly exposed side chains of Ile 16 and Val 17 just below the surface of the enzyme molecule. The finding that Asp 194 is buried in the zymogen as well as in the enzyme is in agreement with the chemical modification studies of Ciarraway et al. (244). 23. D. M.Blow, Biochem. J . 112,261 (1969). 24. K.L. Carraway, P. Spoerl, and D. E.Koshland, J M B 42, 133 (1969).
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J. KRAUT
F I ~4.. Chymotrypsinogen-stereoscopic projection of amino acid residues involved in the formation of the Ile 1bAsp 194 ion pair during activation. The figure is drawn from a vantage point above the surface looking toward the interior of the molecule (from top to bottom on Fig. 2). Figures 4 through 9 were prepared from computer-plotted stereoscopic projections.
B. ARGININE145 The most obvious result of the conformational change in segment V is to move the guanidinium side chain of Arg 145 away from the neighborhood of His 40,Asp 194,and the catalytic site Ser 195 and out into the surrounding medium. This movement is clearly evident upon comparison of Figs. 6 and 7. I n the zymogen, the guanidinium group is resting on the surface of the molecule and may be close enough to the buried side chain carboxylate of Asp 194 to permit appreciable electrostatic interaction between these two oppositely charged groups. Possibly this electrostatic interaction helps to stabilize the structure. I n the enzyme, on the other hand, segment V is rearranged so that the a-carbon of Arg 145 has moved by 9A, and its side chain has swung up to become fully extended into the solvent. That a positively charged side chain in this location may play
5.
CHYMOTRYPSINOGEN : X-RAY STRUCTURE
177
0 -
FIG.5. a-Chymotrypsin-stereoscopic projection of amino acid residues involved in the formation of the Ile 16-Asp 194 ion pair during activation. The figure ia drawn from a vantage point equivalent to that of Fig. 4.
FIQ.6. Chymotrypsinogen-stereoscopic projection of the latent catalytic site region drawn from a vantage point looking into the page (see Fig. 2) at an angle inclined about 20" to the right of perpendicular.
178
J. KRAUT
0 0
cmm OXYGEN NITROGEN
0
p
ASP 102
SULFUR
ASP 102
FIQ.7. a-Chymotrypsin-stereoscopic projection of the catalytic site region drawn from a vantage point equivalent to that of Fig. 6.
some role in the activation process is consistent with the fact that the corresponding residue is also an arginine in pig elastase and bovine thrombin or a lysine in bovine trypsinogen and chymotrypsinogen B (26,26).It must be cautioned before drawing any final conclusions, however, that the electron density map in the region of the Arg 145 side chain is somewhat ambiguous for the zymogen and that an alternative model can be constructed with the side chain extending outward. However, the electron density in the alternative position is much weaker. Perhaps this indicates some degree of disorder with the side chain actually occupying two different positions. In any case, it is difficult to imagine 25. M. 0. Dayhoff, ed., “Atlas of Protein Sequence and Structure” Vol. 4, p. DB4. Natl. Biomed. Res. Found., Silver Spring, Maryland, 1969. 26. S. Magnusson, in “Structure-Function Relationships of Proteolytic Enzymes” (P. Desnuelle, H. Neurath, and M. Ottesen, eds.), p. 138. Munksgaard, Copenhagen, 1970.
5.
CHYMOTRYPSINOGEN : X-RAY STRUCTURE
179
a role for this conformational change in segment V given the alternative position for the Arg 145 side chain.
C. METHIONINE 192 Although segment VI in Table I includes only Met 192 and Gly 193, the conformational change represented here is of considerable interest since it is the only one to which a fairly clear-cut role in the genesis of enzymic activity can be assigned. It results in movement of the side chain of Met 192 from a deeply buried position in the zymogen out to the surface of the enzyme molecule where it forms the flexible hydrophobic lid of the specificity cavity (27). This movement is most clearly visualized by comparing Figs. 6 and 7 but may also be seen in the other figures as well. In the process of moving Met 192 out to the molecular surface, segment 187-193 of the main chain also becomes more extended. The consequence of these combined conformational changes is the creation of the specificity cavity, one side of which is comprised of residues 189192 (27). In the zymogen this cavity does not exist, or a t least it is incomplete and severely distorted. The molecular contortions involved here may be thought of in a simplified way as resulting from a rotation of the main chain about the carbonyl-carbon to a-carbon bond of Asp 194. This rotation causes the shift ,in position of the Asp 194 side chain from the vicinity of His 40 toward the buried N-terminus of Ile 16, as described above, and simultaneously it also moves the backbone chain carbonyl oxygen of Gly 193 into position to accept a hydrogen bond from Nr2 of His 40,replacing the carboxylate oxygen of Asp 194. Thus, the conformational changes involving Ile 16, Met 192, Gly 193, and Asp 194 and formation of the specificity cavity during zymogen activation can be viewed as a single unified event. An attempt has been made to depict certain aspects of the specificity cavity formation from a third vantage point in Figs. 8 and 9. I n Fig. 9, the tosyl group occupying the specificity cavity in tosyl-a-chymotrypsin is shown in black.
D. CATALYTIC SITE As described in the chapters by Blow and by Hess on chymotrypsin, a mechanism for the bond breaking step in the catalytic pathway has been proposed which employs a “charge relay system” (27,28) involving a hydrogen bond network between the side chains of Ser 195, His 57, and Asp 102. It has been very surprising therefore, to find that the spatial arrange27. T.A. Steitz, R. Henderson, and D. M. Blow, J M B 46, 337 (1969). 28. D.M.Blow, J. J. Birktoft, and B. S. Eartley, Nature a21, 337 (1969).
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FIQ.8. Chymotrypsinogen-stereoscopic projection of the latent specificity cavity drawn from roughly the same vantage point as Fig. 2.
F’IQ.9. Tosyl-cr-chymotrypsin-stereoscopic projection of the specificity cavity drawn from a vantage point equivalent to that of Fig. 8. The tosyl group is drawn with solid bonds to contrast with the bonds of the amino acids forming the specificity cavity.
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ment of these three residues in the zymogen is almost indistinguishable from that in the catalytically active enzyme. The term “almost indistinguishable” has been used here because, in fact, a very slight twist may actually occur in the His 57 side chain, and indeed such a twist may be seen by careful comparison of Figs. 2,6, and 8 with the corresponding Figs. 3,7, and 9. If this effectis real, it may suffice to explain the total inactivity of chymotrypsinogen since it would result in distortion of the hydrogen bond network referred to above. Wang (2%) has argued convincingly that facilitated proton transfer along precisely aligned hydrogen bonds may play a crucial role in enzyme catalysis generally. One must be careful, however, to emphasize that the apparent twist in the His 57 side chain is so small that it could well turn out to be spurious when the zymogen electron density map is refined or when direct comparison of the two maps is carried out. Indeed, other subtle but significant structural changes in the charge relay system could also be revealed by further improvement in the data. Nevertheless, at the present stage of the X-ray study of chymotrypsinogen, one can say with reasonable certainty that the catalytic site, in contrast with the specificity cavity, is essentially preformed. The question remains, therefore, as to why the zymogen is not catalytically active, though perhaps without the specificity for hydrophobic side chains characteristic of chymotrypsin. Further to pursue this question, it should be pointed out that two minor structural changes which may also help to explain the zymogen’s inactivity are seen upon activation. These occur not in the charge relay system itself but in its immediate neighborhood: (1) the side chain of Ile 99 moves away from the imidazole ring of His 57, and (2) the hydroxyl group of Ser 214 moves into a better position to form a second hydrogen bond with 082 of Asp 102.These will now be described in more detail. The displacement of the side chain of Ile 99 upon activation is quite noticeable and readily seen on comparing Figs. 6 and 7. I n the zymogen, the Ile 99 side chain is in van der Waals contact with the imidazole ring of His 57 and, together with neighboring side chains, completely blocks access of the solvent to His 57-Asp 102 portion of the catalytic site hydrogen bond network. During activation, the Ile 99 side chain rotates approximately 90” about its a-carbon to P-carbon bond and moves away from the His 57 ring. Curiously, the conformation of the main chain does not alter appreciably. Just why, or even whether or not, this change would be required for inducing enzymic activity is open to question. Such an alteration might be expected, if anything, to decrease the hydro29. J. H. Wang, Science 161, 328 (1968).
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phobicity of the region surrounding His 57-Asp 102, which would in fact appear to be a change in the wrong direction. It has been suggested that the hydrophobic environment of the buried Asp 102 enhances the polarization of the charge relay system and makes the reactive Ser 195 oxygen strongly nucleophilic as required by the proposed mechanism (28).Nevertheless, it is noteworthy that a hydrophobic residue (Ile, Leu, or Val) is conserved a t the position corresponding to Ile 99 in bovine chymotrypsinogen B, bovine trypsinogen, bovine thrombin, and pig elastase (26, 6 6 ) . The change a t Ser 214 is more subtle and at the present resolution remains somewhat doubtful. It involves a possible movement of Oy by perhaps 1 A to form a better hydrogen bond with 062 of Asp 102. In itself, this is probably not a sufficiently large apparent movement to be taken seriously. However, there does occur a rather obvious change in conformation of the chain segment 214-217 during activation. In the zymogen, the stretch of main chain running from the a-carbon of residue 214 to the a-carbon of residue 216 is somewhat folded, but it is fully extended in the enzyme. The effect can be observed readily by comparing Figs. 2 and 3. In light of the proposal by Steitz e t al. (27) that the carbonyl oxygen of Ser 214 is involved in productive binding of substrates, one might expect to see this carbonyl group move into binding position as a result of the main chain extension, but such is not the case. The carbonyl group is in about the same orientation in both zymogen and enzyme. On the other hand, there are several observations suggesting that a second serine residue (Ser 214 in the case of chymotrypsin) forms a hydrogen bond with the same buried aspartic side chain oxygen (082 of Asp 102) that is also hydrogen bonded to the catalytic site histidine side chain (N61 of His 57) in serine proteases generally. This point is discussed more fully in Chapter 15. If this view is correct, the second serine hydrogen bond may well be included in the catalytic site hydrogen bond network and play some role in the activity of these enzymes. It is all the more tempting, therefore, to suppose that distortion of this bond in chymotrypsinogen is one of the factors responsible for its lack of activity and provisionally to consider as significant the apparent slight movement of the Ser 214 side chain.
E. ACTIVATION RDFOLDINQ It will be obvious that at least as many questions about the chymotrypsinogen activation process have been raised as have been answered by the observations reported here. Indeed, the central problem of why the
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aymogen is inactive remains, although it seems reasonably certain that the answer will involve some subtle distortions in and around the catalytic site. Perhaps a suitable way to conclude this chapter would be to mention briefly another, and no doubt more difficult, problem that now arises. Why does the simple act of cleaving a single exterior peptide bond between Arg 15 and Ile 16 cause the chymotrypsinogen molecule to refold in the manner observed? The answer is certainly not obvious from inspection of the two models, and we shall have to content ourselves with simply listing those structural changes which probably contribute, either positively or negatively, to the overall net free-energy decrease upon going from the aymogen to the enzyme conformation. (1) The Arg 15-Ile 16 peptide bond is hydrolyzed to give a free -COOand a free -NH,+ group. (2) The free -NH,+ group tucks down inside the molecule where it interacts with the buried -COO- group of Asp 194. (3) As a result of this process two hydrophobic side chains, a t residues 16 and 17, become buried as well. (4) The buried -COO- group of Asp 194 moves away from the His 40 side chain to which it had been hydrogen bonded and is replaced by the backbone carbonyl of Gly 193 which forms a new hydrogen bond with the His 40 side chain. ( 5 ) The positively charged side chain of Arg 145 moves away from the surface of the molecule, where it may interact with the buried COOof Asp 194, and extends out into the surrounding solvent. (6) The buried hydrophobic side chain of Met 192 moves from the molecular interior out to the surface.
Other conformational changes also occur, some of them quite large, as, for example, in segments I, 111, and I V of Table I. But unlike the events enumerated above for which the situation is fairly clear, it has not yet been possible to see how they might contribute to the free energy of refolding.