V. THE RECEPTORS
V.4. Hydrolytic Enzymes
Hydrolytic enzymes catalyze the splitting of substrates by the involvement of water (30). Many mechanisms of action have been proposed which have been reviewed (30,25). At least three distinct steps may be involved in the enzymatic hydrolysis of ester and amide bonds of substrates by hydrolytic enzymes.
The first step represents the adsorption of the substrate on the receptor, the second the liberation of an alcohol or amine, while the third step represents the
liberation of acid and the reactivation of the enzyme according to the general scheme illustrated in Fig. 25.
In studies with chymotrypsin and p-nitrophenylacetate as a substrate, Gutfreund and Sturtevant (40, 41) have found that the first step is extremely rapid, while the second, viz., the liberation of p-N02-phenol and the acetylation of a part of the receptor, is rate limiting. Studies on pH-dependency provide evidence that a serine OH-group or an imidazole group is involved in acetyl
ation. On the other hand, the liberation of acetic acid appeared highly pH-dependent, in the same way as the overall rate of reactions, indicating that an imidazole group of a histidine residue may be involved in the third step.
The various hydrolytic enzymes are more or less specific in their action; they therefore differ in active site to a certain extent, but they have some features in common, as a serine OH-group or a cysteine SH-group, an imidazole group and
(1) E H + R ' — O - C — R ^ w E H R ' — O - C — R
II II ο ο
(2) E H R ' — O - C — R ^ " E — C — R + R ' — O H
II II ο ο
( 3 ) E - C — R + H20 ^ " E H + R - C - O H
II II ο ο
FIG. 2 5 . Consecutive reaction in enzymatic hydrolysis.
a free carboxyl group. The importance of these groups will be discussed in the next section.
V.4.A. T H E C A R B O X Y L G R O U P
The "anionic" or negative site of the receptors of acetylcholinesterase is composed of one or more carboxyl groups (15). In many other hydrolytic enzymes a carboxyl group of aspartic or glutamic acid is a part of the active site (see Table V). These carboxyl groups are dissociated at the physiological pH, so that electrostatic attraction of an ammonium group or other positively charged groups of the substrate is possible. This implies that there is a prevalent accumulation of substrate or inhibitor molecules around the anionic site.
Subsequently, there is a rearrangement of these molecules on the receptor with the involvement of hydrogen bonds and van der Waals' forces. The carboxyl group has to be ionized, as appears from pH-dependency studies (25, 90). As a matter of fact, the basic group in the substrate must also be dissociated (92).
Thus, 2-ethanol-dimethylammonium is a 30-times better competitive inhibitor of esterase than the structurally similar but uncharged isoamyl alcohol (92).
I V . R E C E P T O R T H E O R Y I N E N Z Y M O L O G Y 245 Further evidence for the fact that oppositely charged groups increase attrac-tion and fixaattrac-tion, is provided by experiments of Wilson et al. with a quaternary inhibitor of esterase, neostigmine, and a tertiary inhibitor, physostigmine (94, 95, 96). The competitive inhibition of esterase by physostigmine decreases with increasing pH, while the inhibition by neostigmine is largely independent of the pH.
The anionic or negative site appears to be extremely important for affinity for acetylcholinesterases, while for pseudo-cholinesterases the negative site should be less important (15). Esterase studies of Bergmann and Segal (15) with homologous series of alkyltrimethylammonium salts and polymethylenebis-trimethyl ammonium salts may indicate that the difference in true and pseudo-cholinesterase is accounted for by the existence of one ionized carboxyl group in pseudo-cholinesterase and two in true cholinesterase.
For other hydrolytic enzymes, too, the presence of an ionized carboxyl group is important for the affinity of substrates. In this respect, the experiments of Smith et al. are interesting to note (81, 82). When studying the hydrolysis of 4 different substrates by papain they found that, although the substrates largely differ in molecular properties and, thus, in affinity and turn-over rate, the pH-dependency of the reaction velocity constant lcx is the same for three substrates and differs for low pH for the one substrate, carbobenzoxyglycyl-glycine (CGG), which contains a free carboxyl group. For the other substrates, the lcx value decreases at a pH below 5.5 but increases for CGG with lowering the pH. These data provide strong support for the supposition that the active site of papain contains an ionized carboxyl group. This ionized carboxyl group accounts for the poor interaction of substrates which contain a free carboxyl group.
The negatively charged groups in most hydrolytic enzymes may direct substrate and inhibitors to the active site. Enzymes like acetylcholinesterase, for instance, which transform small molecules, contain a strong negative charge in the active site, whereas, for enzymes which affect large molecules, groups that are more directing with minor influence may exert this function.
The anionic site, especially, has thus an action in receptor occupancy, but it may also be involved in the release of the transformation products (74, 25).
V.4.B. S E R I N E O H - G R O U P
It is an extremely important observation, that, for a large number of hydrolytic enzymes, serine is involved in the catalytic process and is part of the esteratic site in ACh-esterase (2, 3, 25).
Although entirely different in specificity, a part of the specific receptors of different hydrolytic enzymes has a strongly analogous amino acid composi-tion (25). Activated serine is involved in the catalytic process of breaking ester and amide bonds. In addition to hydrolytic enzymes, the active sites of phos-phoglucomutase (54), and even of thrombin (36, 57), have also the same amino
acid sequence (see Table V). Glutamic acid or aspartic acid is situated adjacent to the seryl group. The serine is thus situated in the sequence shown in Fig. 26 for most of the enzymes.
A large number of investigators have been studying the amino acid compo
sition of different hydrolytic enzymes. They chiefly use techniques analogous
Η
Ο ο II I II
II I . 1 1 ο
c ^ o
οII ο
FIG. 2 6 . Amino acid sequence in the active site of some hydrolytic enzymes.
to those described by Cohen for acetylcholinesterase of bovine erythrocytes (23, 24, 66, 67). This procedure makes use of DFP as an irreversible inhibitor of various hydrolytic enzymes. The inhibition by DFP can be prevented by substrates or competitive inhibitors in such concentrations that they occupy all active sites. With this in mind, an esterase solution is treated with DFP in
TABLE V
AMINO ACID SEQUENCE OF SOME HYDROLYTIC ENZYMES
Enzyme Amino acid sequence Reference
ACh-esterase -Glu- Ser-Ala (103)
Pseudo -Ch -esterase -Phe-Gly-Glu- Ser-Ala-Gly (52) Ali-esterase horse liver -Gly-Glu- Ser-Ala-Gly-Gly (51) α -Chy mo try psin -Gly-Asp- Ser-Gly-Gly-Pro-Leu- (66, 67)
Trypsin -Gly-Gly-Asp-Ser-Gly-Pro-Val-Cys- (29)
Thrombin -Gly-Asp- Ser-Gly-(Glu-Ala) (36, 57)
Elastase -Gly-Asp-Ser-Gly (99)
Phosphoglucomutase -Thr-Ala- Ser-Hist-Asp- (100) Alkaline phosphatase -Val-Thr-Asp- Ser-Ala-Ala-Ser- (103)
Subtilisin -Thr-Ser-Met-Ala (101)
Phosphorylase A -Lys-GluNA2-Ileu- Ser-Val-Arg- (98)
the presence of a sufficient substrate concentration. Under these circumstances nonessential groups may react with DFP. After dialyzation, the enzyme is treated with labeled DFP so that now only the active sites react with DFP3 2. The enzyme then is demolished with trypsin or with acid, and the amino acid sequence of the DFP3 2, containing peptide, is determined—for example, by the Erdmann method.
I V . R E C E P T O R T H E O R Y I N E N Z Y M O L O G Y 247 Inhibition by DFP and degradation of the enzyme may provide information about the number of active sites per enzyme molecule. Chymotrypsin appeared to contain one DFP32-reacting-group per molecule (7, 25); for acetylcholin
esterase this number seems to be larger (25).
Serine largely makes up the esteratic site in the receptor. The esteratic site is a region of high electron density and, therefore, nucleophilic attack on the carbonyl group of the substrate is possible.
According to Wilson et al. (91) the hydrolytic process at the esteratic site may occur as illustrated in Fig. 27. For esters, an acylated esteratic site is formed under liberation of the alcohol, while for reactive phosphates such as di-isopropylfluorophosphonate, a phosphorylated enzyme occurs. The reaction
anionic esteratic site site
FIG. 2 7 . The hydrolytic process according to Wilson (91).
cited above runs smoothly at pH 7 but also at lower pH, whereas at pH above 7, deacylation of the enzyme readily takes place. The acylated enzyme is stable at a low pH. The reaction product of chymotrypsin and p-nitrophenylacetate appeared to be acetylchymotrypsin in acid solution (13, 24, 40).
The oxygen atom in serine does not exert a high electron density, so that it seems doubtful whether the serine-OH group, as such, is responsible for the nucleophilic attack of esters and phosphates. For instance, serine itself does not react with DFP in aqueous solution (5). Serine, necessarily, has to be in an activated form. The serine hydroxyl group may be activated by internal hydrogen bonds with an imidazole group located nearby (19,26). An interesting suggestion has been put forward recently by Rydon (74). Serine in the active site should be a part of an Δ 2-oxazoline nucleus as a result of cyclization with
J. Μ . VAN R O S S U M
the peptide bond in the adjacent asparagyl residue (68) (see Fig. 28). Model studies with A 2-oxazoline derivatives actually revealed that a reaction with DFP is possible (74).
Reaction takes place at the nitrogen atom of the ring, which has a high electron density (Fig. 29). If the residues R and R' have electron attracting properties and, consequently, cause weakening of the basic properties of the nitrogen atom in the oxazoline nucleus, reaction with phosphorus compounds
Η FIG. 28. Active serine in hydrolytic enzymes.
is impaired. Rydon (74) put forward the hypothesis that the unique role of serine, in fact, is by means of an oxazoline nucleus. The basic strength of the nitrogen in the ring should be high enough to assure nucleophilic attack on the carbonyl C atom of esters or the phosphoryl Ρ atom of phosphates.
As result of an activated serine group in the active site of hydrolytic enzymes with phosphor compounds an O-phosphoryl-serine enzyme is formed and with esters an O-acyl-serine enzyme.
FIG. 29. Phosphorylation models of active serine.
A detailed description of the sequence of events in the catalytic process has been proposed (11, 20, 25, 74). The suggestion, that the adjacent protonated carboxyl group of an aspartic residue must be essential for the release of the acyl group from the esteratic site, seems untenable. The ipKn value of the carboxyl group is approximately 4, whereas the hydrolysis of acylate chymo-trypsin is controlled by a group having a pifa value of approximately 7.4, which presumably is an imidazole group (19).
V.4.C. T H E S U L F H Y D R Y L G R O U P
In most enzymes a serine OH-group is involved in the active center, but in other ones an analogous function is ascribed to an SH of cystein. Papain, for instance, contains an SH group, essential for its catalytic action upon peptides.
It has recently been suggested that also the SH group in the active center is activated by ring formation with an adjacent peptide bond (75).
I V . R E C E P T O R T H E O R Y I N E N Z Y M O L O G Y 249 Saroff has communicated the role of oxazoline and thiazoline ring-systems in proteins and their implications in protein reactions (75). He suggests that the ring is essential for activity, while the equilibrium between the ring and the open system depends on the pH. The amino acids in the direct vicinity of the ring system, as aspartic acid, lysine, andhistidine, for instance, are responsible for the pH-dependency. On this basis, Saroff was able to explain thepH opti-mum of different enzymes, for instance, why trypsin has an optiopti-mum at pH 2 and other hydrolytic enzymes at pH 7.
V.4.D. T H E I M I D A Z O L E R I N G
Studies on the pH-dependency of various hydrolytic enzymes have revealed that an imidazole nucleus from histidine may participate in the catalytic pro-cess (8, 12,19). Histidine is not located in the amino acid chain in the neighbor-hood of serine, but may be part of other loops of amino acids, which, by folding, have placed the imidazole nucleus in a favorable position. The fact, that deacetylation of an acylated enzyme is accelerated by increasing the pH, points to histidine catalyzed deacylation. Many theories have been proposed as to the mechanism of action of the imidazole nucleus. It may be said, however, that the more accepted role of the histidine residue is that it effects the release of the acyl group of the acylated enzyme. In fact, it acts as a nucleophilic agent held in the correct position by the peptide structures (8, 25).
Since in phosphorylated enzymes the phosphate groups are not so readily released, additional nucleophilic agents are necessary, for instance, hydroxyl-ions. In fact, chymotrypsin inhibited by isopropyl methylphosphonofluoridate (sarin) recovers activity, and this is accelerated by an increase in pH. Other nucleophilic agents, such as oximes and hydroxamic acids, are able to restore activity of inhibited enzyme and are, therefore, called reactivators (91).
V.5. Reactivators
Nerve gases, which are irreversible esterase inhibitors, are phosphorus compounds which contain an easily hydrolyzable phosphor bond. They act by phosphorylation of an essential part of the active site, presumably, the serine OH-group (1) (see Fig. 30). The phosphor bond with the serine OH-group is stable in aqueous solution. Enzymes like chymotrypsin, after irreversible inhibition recover slowly. Regaining of activity is accelerated by hydroxyl ions (38, 39). The OH-ions involve a nucleophilic replacement of the phosphate group in the receptor. Since the OH-ion is very small it can approach the phosphor bond, whereas imidazole in the active site has a less basic strength, and being larger, is unable to do this. Imidazole is able to catalyze the release of an acyl group, since an acyl group is planar and thus easily accessible, while for the phosphoryl group, there is a spatial configuration to keep in mind.
Oximes and hydroxamic acid, by virtue of their nucleophilic properties, are also able to reactivate the active sites (91) (Table VI). With respect to the
T A B L E V I
REACTIVATORS OF HYDROLYTIC ENZYMES INHIBITED WITH ORGANOPHOSPHONATES
Name Code Formula
Hydroxy ions OH O H
-Choline Ch C^+ C—OH
C—N—
C^
Hydroxylamine
Glycolhydroxamic acid AGH
H2N — O H
H O — C \ Q / N — O H ii Η
Nicotine-hydroxamic acid methiodide NHAM / N V / CN - O H Η N
2-Picoline-hydroxamic acid
^ N ^ C ^ g - °H
Η Jl H
Monoisonitrosoacetone
Diacetylmonoxime
ΜΙΝΑ
DAM
Ο
G/ C \C^ N — O H Ο
C ^ C K ^ N — O H C
Pyridine-2-aldoxime methiodide 2-PAM OH
C
1,3 -Bis- (pyridinium-4 -aldoxime) -propane TMB -4 dibromide
:N I OH C — N + V-CL
\ / Ν I OH
IV. RECEPTOR THEORY IN ENZYMOLOGY 251 reactivation of chymotrypsin inhibited by sarin, various hydroxamic acids and oximes are equally active. This is in line with their equal reactivities to sarin itself (25). With respect to reactivation, however, of cholinesterase, there
anionic esteratic site site
FIG. 3 0 . The phosphorylation of the esteratic site by an organophosphonate.
are large differences in potency (25). Hydroxylamine is able to reactivate esterases but, for this, a large dose is required (96). The process of reactivation is given in Fig. 31 and the kinetics has been discussed by Davies and Green (28).
anionic esteratic site site
C
FIG. 3 1 . The process of reactivation by PAM (96).
The high degree of complementarity of reversible cholinesterase inhibitors, stressed by Wilson, does logically lead to the expectation that, for reactivation also, complementarity is an essential factor (94, 96).
Planar aromatic rings contribute highly to binding with the receptor.
Moreover, a quaternary ammonium group with a positively charged nitrogen
atom in the molecule will contribute to affinity, especially to the anionic site (96). With some idea of the distance of the esteratic and anionic site, Wilson could expect that 2-pyridine-aldoxime methiodide (2-PAM) would be a potent reactivator of inhibited esterase. This indeed appeared to be exactly the case.
It has been found that di-quaternary ammonium salts are better as rever
sible cholinesterase inhibitors than the mono-quaternary ones. Reactivators have been developed with two N+ atoms with this in mind, for instance, by quaternization of pyridine aldoxime with a, ω-dibromodecane (#0). Reactiva
tion also depends on the nature of the irreversible inhibitor. It is more difficult to reactivate acetylcholinesterase inhibited with DFP than when inhibited with its dimethyl derivative (25). Steric hindrance accounts for this difference.
Irreversibly inhibited cholinesterase slowly changes on storage from a form which can be reactivated by nucleophilic agents into a form which cannot.
Aldridge has suggested the explanation that, on storage, one of the alkyl groups is split off from the alklylphosphoryl residue under formation of phosphate anion, which is resistant to nucleophilic attack by a reactivator (3).
anionic esteratic
RO bR RO OH + ROH FIG. 32. The process of "aging" («57, 52).
Dealkylation is promoted by the anionic site in esterase, while, in chymotrypsin, such an interaction is not so easily performed, owing to the lack of an anionic site.
Recent studies have shown that a DFP-inhibited pseudo-cholinesterase can be reactivated by oximes, whereas on storage one isopropyl group is released from the inhibited enzyme (14, 24). The diisopropylphosphate enzyme, thus, on "aging" slowly converts to a monoisopropylphosphate enzyme, as shown in Fig. 32. Analogous reactions may be the cause of a failure, in general, of oximes to reactivate the irreversibly inhibited enzymes (14, 25).
CONCLUSION
The contact of chemical substances with living organisms are on the molecular level. There is, therefore, a strong analogy between: the action of substrates and inhibitors on the active site of enzymes; the action of chemical stimuli on sensory receptors; and the action of drugs on receptors of every tissue in the body.
When studying drug actions in man or animals enzymatic reactions and drug-receptor interactions are often simultaneously involved. A drug may
IV. RECEPTOR THEORY IN ENZYMOLOGY 253 cause an effect after being converted into certain products by specific enzymes while these products may cause a release of endogenous substances which in turn evoke the effect. It is therefore important for the pharmacologist to have some knowledge of other fields of molecular biology.
Throughout these volumes there is found a similar approach to the problems, and in this chapter, a general discussion is given on the interaction of substrates and other substances with the active site. In addition, recent advances in the elucidation of the active site of some enzymes have been discussed. The depth of the approach to the receptor in enzymology is not possible in other fields but advances in one field may be very fruitful and stimulating for other fields of biological sciences.
REFEBENCES 1. W . N. Aldridge. (1953). Biochem. J. 55, 763.
2. W . N. Aldridge. (1954). Chem. ώ Ind. 473.
3. W . N. Aldridge. (1957). Ann. Repts. on Progr. Chem. 53, 294.
4. E. J. Ariens. (1954). Arch, intern, pharmacodynamic 99, 32.
5. R. F. Ashbolt and Η. N. Rydon. (1957). Biochem. J. 66, 237.
6. Κ. B. Augustinsson and D. Nachmansohn. (1949). J. Biol. Chem. 212, 543.
7. A. K. Balls and E. F. Jansen. (1952). Advances in Enzymol. 13, 321.
8. E. A. Barnard and W . D. Stein (1958). Advances in Enzymol. 20, 51.
9. H. Baum, G. Hubscher, and H. R. Mahler. (1956). Biochim. et Biophys. Ada 22, 514.
10. R. F. Beers, Jr. (1955). J. Phys. Chem. 59, 552.
11. M. L. Bender and B. W . Turnquest. (1957). J. Am. Chem. Soc. 79, 1652.
12. M. L. Bender and B. W . Turnquest. (1957). J. Am. Chem. Soc. 79, 1656.
13. L. Benoiton, Η. N. Rydon, R. A. Oosterbaan, Μ. E. van Adrichem, and J. A. Cohen.
(1960). Nature 187, 596.
14. F. Berends, C. H. Posthumus, I. van der Sluys, and F. A. Deierkauf. (1959). Biochim.
et Biophys. Acta 34, 576.
15. F. Bergmann and R. Segal. (1954). Biochem. J. 58, 692.
16. P. D. Boyer, H. Lardy, and K. Myrback, eds. (1959). " T h e Enzymes," Vol. I. Aca
demic Press, New York.
17. G. E. Briggs and J. B. S. Haldane. (1925). Biochem. J. 19, 338.
18. W . Brock Neely. (1958). J. Am. Chem. Soc. 80, 2010.
19. D. M. Brouwer. (1957). Ph.D. Thesis. University of Leiden, Netherlands.
20. T. C. Bruice and G. L. Schmir. (1957). J. Am. Chem. Soc. 79, 1663.
21. B. Chance. (1955). Advances in Enzymol. 12, 152.
22. J. A. Cohen, M. G. P. J. Warrihga, and B. R. Bovens. (1951). Biochim. et Biophys.
Acta 6, 469.
23. J. A. Cohen, R. A. Oosterbaan, and M. G. P. J. Warringa. (1955). Biochim. et Biophys.
Acta 18, 228.
24. J. A. Cohen, R. A. Oosterbaan, H. S. Jansz, and F. Berends. (1959). J. Cellular Comp.
Physiol. 54, 231.
25. J. A. Cohen and R. A. Oosterbaan. (1962). In "Handbueh der experimentellen Pharmakologie," (A. Heffter and H. Heubner, eds.), Vol. 15, ρ .299. Springer, Berlin.
26. L. W . Cunningham. (1957). Science 125, 1145.
27. D. D. Davies and E. Kun. (1957). Biochem. J. 66, 307.
28. D. D. Davies and A. L. Green. (1956). Biochem. J. 63, 529.
29. G. H. Dixon, D. L. Kauffman, and H. Neurath. (1958). J. Biol. Chem. 233, 1373.
30. M. Dixon and E. C. Webb. (1958). "Enyrnes." Academic Press, New York.
31. K. S. Dodgson, B. Spencer, and K. Williams. (1956). Nature 177, 432.
32. G. S. Eadie. (1942). J. Biol. Chem. 146, 85.
33. L. H. Easson and E. Stedman. (1936). Proc. Roy. Soc. B21, 142.
34. B. Ellenbroek and J.M. vanRossum. (1960). Arch, intern. Pharmacodynamic 125, 216.
34a. Ε. H. Fisher, D. J. Graves, E. R. Snijder-Crittender, and E. G. Krebs. (1959). J. Biol.
Chem. 234, 1698.
35. R. F. Furchgott. (1955). Pharmacol. Revs. 7, 183.
36. J. A. Gladner and K. Laki. (1938). J. Am. Chem. Soc. 80, 1263.
36. J. A. Gladner and K. Laki. (1938). J. Am. Chem. Soc. 80, 1263.