California Association of Chemistry Teachers
B. R. Baker
Interactions of Enzymes and Inhibitors
University of California Sonto Borboro
T h e kinetic parameters of an enzyme (E) catalyzed conversion of substrate (S) t.o product (P) are expressed in eqn. (1). The first st,ep is a fast diffusion E+S
a
E...S
1:
a E.,.P
i3
E+P
(1)
n 1+x-
E + I-X
E
(3)
The second type of irreversible inhibitor is expressed in eqn. (4).
Km
process expressed hy the equilibrium constant, K,. The rate determining step of the process is expressed by k. Suchaprocessrequires that theenzyme and substrate form a reversible complex, E . . .S. Certain changes in S can lead to a molecule which still can form a reversible complex with theenzyme but whichis not converted to a product; such a molecule will therefore be a reversible inhibitor (I) of the process by format,ion of a11 E . . .I complex, as expressed in eqn. (2) E+I
a
E...I
K*
(2)
the equilibrium constant being K,. Two of the subjects of this presentation are the possible nature of the binding forces between enzyme and inhibitor or substrate and the specific groups on the inhibitor which are required for binding to selected specific enzymcs. A second kind of enzyme inhibition is t,he irreversible type due to formataimof a covalent bond between the enzyme and the inhibitor. Enzymes are large polypeptides made up from twenty different amino acids; those amino acids having a third functional group such as the sulfide of methionine, the imidazole of histidine, t,he hydroxyl of tyrosine, etc., that happen t,o be on the surface of the macromolecule can be attacked by chemical reagents with formationof a covalent linkage. If t,his new covalent linkage interferes with the subsequent formation of an E . . .S complex, or interferes with t,heability of the enzyme to convert the substrate to t,he product within the complex, t,hen t.he enzyme will have been inhibited irreversibly. There are two main types of irreversible inhibitors. The first type of irreversible inhibitor reacts with an essential functional group on the enzyme by a bimolecular process (eqn. (3)), which has litt,le specificity since all groups on the surface of all enzymes, with t,he nucleophilic capacity to do so, will react a t varying rates expressed by ko. Presented at, tthe CACT Conference, Ssnta Barbara, Calif., December, 1066. Paper 96 in n series on Irreversible Enzyme Inhil,it,ors.
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The enzyme forms a reversible complex with the inhibitor bearing a leaving group, X. If the leaving group, X, and a nucleophilic group on the enzyme are closely juxtaposed, then a rapid neighboring group reaction with formation of a covalent bond will occur within the complex. Such a reaction can be highly specific since properly juxtaposed neighboring groups can interact 1000-10,000-fold faster than the corresponding bimolecular reaction, that is, k , for a directed irreversible inhibition is 3-4 maguitudes more rapid than kb for a bimolecular reaction. Thus the concept of active-sitedirected irreversible enzyme inhibition, (eqn. (4)) emerged (1-5) mhich can be stated as follows: "The macromolecular enzyme has functional groups on its surface mhich logically could be attacked selectively in the tremendously accelerated neighboring group reactions capable of taking place within the reversible complex formed between the euzyme and an inhibitor snbstituted with a properly placed neighboring group." Binding Forces Between Enzymes and Other Molecules
The forces bet,ween an enzyme and inhibitor or substrate that allow complex formation can be divided into two generalized classes. The first class consists of complexes between electron donors (D) and electron accept,ors (A) (4) ; t,heelectron donor group may be on the enzyme, as in eqn. ( 5 ) ,or on theinhibitor, as in eqn. (6). E-ll+ A-I
E--A
+ D-I
KA
e
K*
E-D
a E-A
-
A-I
(5)
D-I
(6)
The force of the bonding is expressed by the equilibrium const,ant, K A . In this class are (a) coulomhic (anionic cationic) interact,ion, (b) hydrogen bonds, and (c) charge-tmnsfer complexes (5, 6). Since in each case there is a donor and acceptor partner, it is possible t o have mived types; for example, an electron-rich a-cloud
can be a donor to a hydrogen which has the electron de-
Although the bonding energies in kcal/mole of these donor-acceptor complexes have been measured in inert solvents, these measurements represent maximal values, since the binding to enzymes occurs in water where water molecules can also form donor-acceptor complexes (4). The second class of complex formation involves bydrophobic bonding and the accompanying van der Waals forces;this isshown diagrammatically in Figure 1, where the circles represent water. The solution of the
Figure 1.
Hydrophobic bonding.
hydrocarbon portions of A and B into water requires ellergy due to a change in water structure. When the solutions are mixed, a complex between A aud B molecules can form with regeneration of the origiual water structure; thus part of the energy required to dissolve A and B in water will be released when com~lexformation occurs (4). This process of hydrophobic binding is highly favorable for complex formation and has a maximum free energy release of 0.7 kcal/mole per >CH,. . . CH2< interaction (8,Q).I n addition there call be further affinity of A aud B due to van der Waals forces or dipole-dipole interaction; the van der Waals forces cau also have a maximum bonding energy of 0.7 kcal/moleof methylenemethylene interaction ( 8 , l O ) . Thus simple hydrocarbon bonding could have as much as 1.4 kcal/mole per methylene on the substrate or inhibitor, or about one power of ten in the dissociation constant. When hydrophobic bouding with an inhibitor can be fouud, i t is a most useful phenomenon, as will be discussed later.
S+FX
F;?
SX+F
In order for the group X to be transferred to the substrate, the group X must be held in juxtaposition between F and S by the active site of the enzyme as shown in Figure 2; if more than a single interatomic distance exists betwcen S and X, then new bond formation to form SX will be difficult. If the group X is replaced by B (Fig. 3), an inhibitor resultsif B cannot be transferred. If B is a leaving group that is juxtaposed to a nucleophilic group within the active site, then covalent bond formation will occur which stops dissociation of the enzyme-inhibitor complex, and the active site becomes selectively denatured; since this reactiou occurs inside the active site it is called active-site-directed irreversible iuhibition by the endo mechanism (1-3). This endo mechanism has two serious drawbacks, namely (a) the B group must be no larger than X or i t cannot fit within the active site, and (b) the identical enzyme from differeut species or even mechanistically related enzymes will also be iuactivated by FB. Exo Mechanism. If the leaving group B (Fig. 4) is on a side of the inhibitor uot in contact with enzyme and is placed so that it cau bridge back to some nucleophilic group on 1,heenzyme surface outside the active site, then covalent bond formation could take place (shown by arrow) (1-5). The irreversible inhibition by the exo mechanism overcomes the objections inherent in the endo mechanism since (a) large groups can be used between A aud B, and ( b ) it is outside the active site where evolutionary changes of amiuo acids can occur most readily-that still retain a function enzymeresulting in the possibility for differential irreversible inhibition of the identical euzyme from different tissues or species (11). Lactic Dehydrogenase
Lactic dehydrogenase is an enzyme that oxidizes lactate to pymvate mediated by DPN or reduces pymvate to lactate with DPKH (eqn. (8)). C H ~ C H C O OC~H ~ C C O O ~
II
I
OH
x0
DPN
(8)
DPNH
Types of Active-Site-Directed Irreversible Enzyme Inhibitors
Zndo Mechanism. Enzymatic reactions utiliziug a cofactor can be written in the general form of eqn. (7).
m, Figure 2. A rimpliRed diagram of the enzymecatalyzed transfer of the group X from o sofoctor F to the substrate 5, both molecules being jvxtoporitioned at the active site of the enzyme. From reference(21.
Figure 3. A rimpliRed diogram of endoallylotion. A group, B, replaced the trowfer group X in Figure 1. B son then olkylote mme nucleophilic group within lend01 the active site to form o covalent bond. From reference (2).
Figure 4. A simplified diagram of an ovenire (noncla~sisol) inhibitor. Note that the excess sire above the horizontal dotted line of substrate faces away from the enzyme surface. When the aikylating group B can bridge to o nvcleophilic rite on the enzyme surface adjacent to the octive $te, covalent bond formotion lorrow) occur, outside the active site ( e m olkylotionl. From reference (21.
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The main binding by lactate is through its caborxylate and hydroxyl groups. This binding can be simulated by the o-hydroxyl and carboxylate groups of salicyclic acid; since the carbon of salicylate hearingthe hydroxyl group does not also bear a hydrogen for transfer to DPN, salicylate is a reversible inhibitor. Salicylate can he converted to an irreversible inhibitor of the exo-type by placement of a function on the benzene ring that has a leaving group (Fig. 5).
-Skeletal Comod.
Moscle LDHKI Rate of 1mMi Inactivstion
Figure 5. Selective irreversible from different tisuer.
-Heart LDFIKI Rate of imMi Inaetmrsl~on
inhibition of
lactic dehydrogenase
4-(1odoacetamido)salicylic acid (I, Fig. 5) forms a reversible complex with lactic dehydrogenase from either skeletal muscle or heart; however, only the skeletal muscle enzyme is irreversibly inhibited by I (Fig. 5) with a half-life about 30 min (11, 13). When the distance between the carboxylate and the leaving group is lengthened as in I1 (Fig. 5), then both enzymes are irreversibly inhibited; the heart enzyme is inactivated with a half-life of about 60 min (11, IS). When the leaving group is changed to pbenoxy, at the 4- or 5-position (Structures I11 and IV, respectively), both enzymes are reversibly inhibited about equally by both compounds; however, only I11 (Fig. 5) inactivates the skeletal muscle enzyme and only IV (Fig. 5) inactivates the heart enzyme-a crossover in specificity (11, 14). Since reversible inhibition still allows the enzyme to convert substrate to product, but irreversible inhibition gives a non-functional enzyme, only the irreversible inactivation is important with an inhibitor of the type in Structures I-IV given in Figure 5. Dihydrofolic Reductase
Dihydrofolic reductase is an enzyme that can convert eitherfolicacid I1 (aB-vitamin) or dihydrofolic acid I11 to the cofactor form, tetrahydrofolate IV. There are two main classes of inhibitors of this enzyme (15, 16). 612
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The first class are close analogs where the 4-0x0 group of folic acid, (II), has been replaced by amino, as in V and VI; both V and VI are extremely potent inhibitors of dihydrofolic reductase and VI, known as methotrexate, is used for treatment of leukemia. The second class does not contain the p-aminobenzoyl-L-glutamate moiety of folic acid 11,but has instead a hydrocarbon like moiety; for example, VII is a good antimalarial agent known as Daraprim and VIII is a good antibacterial agent known as trimethoprim. The hydrocarbon groups at Ra are complexed to a hydrophobic bonding region of dihydrofolic reductase that is adjacent to the active site, but not part of the active site; evidence has been found that this hydrophobic bondiug region is adjacent to the area where the4-oxogroup of folic acid (11) resides on the enzyme (16-18); thus the pyrimidine of VII and VIII is shown with the R5 group in the area where the &ox0 group is shown in 11,when they are complexed to the enzyme.
Dihydrofolic Acid
Tetratiydrofolic Acid
V,R-H VI, R = CH,
&&HZ N v N NH2
VII : RS=p-ClCsHa; Rs = CaHs VIII: RS=3,4,5-(CHaO)aCsHGH2; Ra= H
Hydrophobic Bonding. Since this hydrophobic bonding region is not part of the active site, it can be anticipated that evolutionary changes would have occurred in this region; therefore one should expect to observe differencesin the ability of hydrocarbon groups to bind to this region as enzymes from various species are examined. Some comparisons are shown in Figure 6 with the dihydrofolic reductases from pigeon liver, E. coliB, and the enzymeinduced by the bacterial virus, T,phage, when it infects E. coli B.
No.
R
V Trimethoprim
pM Conc. for 50y0 Inhibition Pigeon Tr E . eoli Liver Phage B
16
0.68
0.0003
All assays with 6 rmM dihydrofolate.
Figwe 6. Species differences in hydrophobic bonding to dihydrofolic reductare b y the given structures.
The results in Figure 6 were selected from some fifty compounds that were assayed (19). Note that that phenyl binding (I in Fig. 6) with the Trphage enzyme is similar to pigeon liver enzyme but different from E. coli B enzyme. Even more striking are the results with the antibacterial agent, trimethoprim (V in Fig. 6), which has previously been shown to be much more effective on bacterial dihydrofolic reductases than the mammalian enzymes (20, 91). Note that structure V (Fig. 6) complexes 50,000-fold better to the dihydrofolic reductase from E. coli B than the enzyme from pigeon liver and 2300-fold better than to T2-phage iuduced enzyme. In contrast there is only a 23-fold difference in binding to the pigeon liver enzyme and the Tz-phage induced enzyme. However, there are some similarities in common with the E. coli B and Trphage induced enzymes such as (a) the p-phenyl substitutent of structure I1 (Fig. 6) causes a 1400-fold loss in binding to the pigeon liver enzyme, but only a 2-fold and an 8-fold loss, respectively, to the E. coli B and Tz-phage iuduced enzymes; (b) the m-phenyl substituent of structure 111, (Fig. 6) gave a 3-fold and 14-fold better binding to the E. coli B and T2-phage induced enzymes, respectively, but a 12-fold loss in binding to the pigeon liver enzyme. The results in Figure 6 give considerable insight
into the evolutionary differences in preferred conformations of hydrocarbon groups for binding to this hydrophobic region (16). The T,phage induced enzyme has some striking similarities to a vertebrate enzyme such as that from pigeon liver; however, there are also similarities between the Tzphage induced enzyme and the host bacterial enzyme. These results have some important hearing on the time in evolution when the T,-phage emerged; pigeon and Trphage diverged from a common ancestor much later on the paleontological time scale than E. coli B, and pigeon diverged from a common ancestor. Such time sca'e divergences are more accurately done by determination of linear sequences of amino acids of the pure enzyme from different sources (22, 23); such studies with dihydrofolic reductase have the additional difficultythat only 0.001-0.005% of the total cellular protein consists of dihydrofolic reductase; it is therefore difficult to obtain sufficient amounts of pure dihydrofolic reductase for amino acid sequence studies. Hitchings, et al. (21) have been fabulously successful in finding 2,4-diaminopyrimidines with varying hydrocarbon groups such as VII that are successful agents for treatment of bacterial and protozoan diseases; it is highly probable that the selectivity of action is due to the 50,000-fold or more differences in binding to the hydrophobic bonding region of dihydrofolic reductase where evolutionary changes had occurred. Such large differences would not be expected among the tissues of an animal. The largest differences we have observed (19) in hydrophobic bonding to dihydrofolic reductase from different mammalian tissues has only been 100-fold; somewhat larger differences might be anticipated with a tumor inducing virus, but eveu these differences will probably not be large enough to exploit with reversible inhibitors. Active-Site-Directed Irreversible Inhibition. It should be possible to greatly magnify small differences in the hydrophobic bonding- region of dihydrofolic reductases from tumor and normal tissues~bybringing in the extra parameter of active-site-directed irreversible inhibition; such an approach is shown in Figure 7 (24). The maximum specificity should be observed
a
I' Figure 7. Schematic representation of an active-sibdirected irreversible inhibitor that utilizer hydrophobic bonding for specificity; AS = Active site binding of on inhibitor; HP = hydrophobic binding of on inhibitor; and Nu = an enzymic nucleophilic group.
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if an alkylating function on the inhibitor can he bridged hack from the hydrophohic region on the enzyme to the more polar nucleophilic region. In this way the ability of an irreversible inhibitor to bridge between the pyrimidine locus in the active site and the enzymic nucleophilic site can he controlled by the nature of the hydrophobic site, which is outside the active site and where the greatest evolutionary diierences can occur (11). It should also he possible to oht,ain specificity by varying the group that complexes in the hydrophobic region; this could alter the position of the alkylatimg function (dot,ted arrow, Figure 7) by positioning the pyrimidine slightly differently; this can be likened to a fulcrum where a slight shift on the hydrophobic side will shift the alkylating side in the opposite direction where a part of the pyrimidine is the axis of the fulcrum.
reversible inhibition is the same with both enzymes. Compare the structure in Figure 8 with the diagram in Figure 7 where the hromoacetamido group is part of the dotted line. If the chloropheuyl group in Figure 8 is changed to phenylbutyl, reversible binding to both enzymes is still excellent, hut now neither enzyme is irreversibly inhihited-demonstrating the fulcrum principle above. Better irreversible inhibitors soon emerged (26) as shown in Figure 9 with the enzyme from mouse L-1210 leukemic cells. The compound in Figure 9 could inactivate the leukemic enzyme with a halflife of about 5 miu in the presence of TPNH. Similarly, the enzyme from the Walker 256 tumor from the rat and the enzyme from rat liver could he inactivated with a half life of < 2 min. In contrast, the enzyme from pigeon liver was inactivated more slowly. In the absence of TPNH, the rat tumor enzyme was inactivated with a half life of less than 30 sec. Synthesis of related compounds to build in more selectivity-by utilization of principles previously outlined (2, 11)-into irreversible inhibitors related to the compound in Figure 8 are underway; i t is within the realm of practicality to obtain compounds which, by the active-site-directed mechanism, will inactivate the enzyme from a tumor such as the Walker 256 with mineral irreversible inhibition of the enzyme from normal rat tissues. Hydrophobic Bonding to Other Enzymes
K , X 106M uM Cone.
Source
Pigeon liver E. eoli
0.09 0 1
0.25 0.1
%
%.
E...I
Inactivat,ion
80 50
0 33
Enzymes that have been investigated for hydrophobic bonding in this laboratory are listed in Figure 10. If a hydrophobic honding region adjacent to the active site can be found, it is a most useful phenomenon -as described in the previous section. In not all cases can a hydrophohic bonding region be found; for example, an extensive search on thymidine kinase failed to reveal such a hydrophobic region (27, $8). No useful hydrophohic region has yet been found on thymidylate synthetase, but not all areas on the inhibitor have been investigated (29). Although weak hydrophobic honding to sucdnoadenylate kinosyn-
Incubated 2 hr with 30 uM TPNH at pH 7.4 and 37" C. Figure 8. Selective inhibition of dihydrofolic reductass from differsnt specie. by the obove compound.
Demonstration of these predictions (24) soon followed. In Figure S is shown (25) selective irreversible inactivation of B. coli B dihydrofolic reductase without inactivation of the pigeon liver enzyme; note that,
= He,.
N@CI
N H AR~ K I X 106 M
fiM Conc. Inhibitor
E...I
H~~.
(33) ~ e i i e f e k n e e(3), chapters V and VII. (34) BAKER,B. R., J . Med. Chem., 10, 69 (1967).
(35) BAKER,B. R., AND WOOD,W. F., lo, Nov. (1967). H. M., J. Biol. Chem., 167, 429 (1947). (36) KALCKAR, (37) BERGMANN, F., LEYIN, G., KWIETNY-GOURIN, H., AND UNGAR, H., Biochim. Biophys. Acla, 47, 1 (1961). J. L., J. Pharm. S&., 56, (38) BAKER,B. R., AND HENDRICKSON, in press (1967). S , H., Ann. N . Y . (39) ELION,G. B., BIEBER,S., AND ~ ~ I T C H I N GG. A d . S&., 60, 297 (1954). J.,J.Med.Chern., 10,682(1967). (40) BAKER,B. R., AND KOZMA, (41) BAKER,B. R., J. Pharm. Sei., 56, in press (1967). (42) BAKER,B. R., Ho, B.-T., SANTI,D. V., J . P k a m . Sei., 54, 1415 (1965). G. J., J . Pharm. (43) BAKER,B. R., Ho, B.-T., AND LOURENS, Sei., 56, 737 (1967). Volume
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