interaccion de enz. - inh

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California Association of Chemistry Teachers

The kinetic parameters of an enzyme (E)catalyzed conversion of substrate (S) t.o product (P) areexpressed in eqn. (1). The first st,ep is a fast diffusion

B. R. BakerUniversity of California

Sonto Borboro

1E + S a E . . .S a E . , . P i3 E + P ( 1 )Kmprocess expressed hy the equilibrium constant, K,.The rate determining step of the process is expressed byk. Suchaprocessrequires that theenzyme and substrateform a reversible complex, E . ..S. Certain changes inS can lead to a molecule which still can form a reversiblecomplex with theenzyme but whichis not converted to aproduct; such a molecule will therefore be a reversibleinhibitor (I) of the process by format,ion of a11 E . . Icomplex, as expressed in eqn. (2)

E + I a E . . . IK* ( 2 )

Interactions of Enzymes and Inhibitors

the equilibrium constant being K,. Two of the subjectsof this presentation are the possible nature of the bindingforces between enzyme and inhibitor or substrate andthe specific groups on the inhibitor which are requiredfor binding to selected specific enzymcs.A second kind of enzyme inhibition is t,he irreversibletype due to formataimof a covalent bond between theenzyme and the inhibitor. Enzymes are large polypep-tides made up from twenty different amino acids; thoseamino acids having a third functional group such as thesulfide of methionine, t he imidazole of histidine, t,hehydroxyl of tyrosine, etc., th at happen t,o be on the sur-face of the macromolecule can be attacked by chemicalreagents with formationof a covalent linkage. If t,hisnew covalent linkage interferes with the subsequent for-mation of an E . . S complex, or interferes with t,he abil-ity of the enzyme to convert the substrate to t,he productwithin the complex, t,hen t.he enzyme will have been in-hibited irreversibly.There are two main types of irreversible inhibitors.The first type of irreversible inhibitor reacts with an es-sential functional group on the enzyme by a bimolecularprocess (eqn. (3)), which has litt,le specificity since allgroups on the surface of all enzymes, with t,he nucleo-philic capacity to do so, will react at varying rates ex-pressed by ko .Presented at, tthe CACT Conference, Ssnta Barbara, Calif.,December, 1066. Paper 96 in n series on Irreversible EnzymeInhil,it,ors.

nE + -X E 1 + x - (3)The second type of irreversible inhibitor is expressedin eqn. (4).

The enzyme forms a reversible complex with t he inhibi-tor bearing a leaving group,X. If the leaving group,X,and a nucleophilic group on the enzyme are closelyjuxtaposed, then a rapid neighboring group reactionwith formation of a covalent bond will occur within thecomplex. Such a reaction can be highly specific sinceproperly juxtaposed neighboring groups can interact1000-10,000-fold faster than th e corresponding bimolec-ular reaction, tha t is, k , for a directed irreversible inhi-bition is 3-4 maguitudes more rapid than kb for a bi-molecular reaction. Thus the concept of active-site-directed irreversible enzyme inhibition, (eqn. (4))emerged (1-5) mhich can be stated as follows:"The macromolecular enzyme has functional groupson its surface mhich logically could be attacked selec-tively in the tremendously accelerated neighboringgroup reactions capable of taking place within the rever-sible complex formed between the euzyme and an inhib-itor snbstituted with a properly placed neighboringgroup."Binding Forces Between Enzymes andOther Molecules

The forces bet,ween an enzyme and inhibitor or sub-strate tha t allow complex formation can be divided intotwo generalized classes. The first class consists of com-plexes between electron donors (D) and electron accep-t,ors (A) (4) ; t,heelectron donor group may be on the en-zyme, as in eqn. ( 5 ) ,or on theinhibitor, as in eqn. (6).

KAE- l l + A-I e E-D - -I ( 5 )K*E--A + D-I a E-A - -I (6)

The force of the bonding is expressed by the equilibriumconst,ant,KA . In this class are (a) coulomhic (anioniccationic) interact,ion, (b) hydrogen bonds, and (c)charge-tmnsfer complexes (5, 6). Since in each casethere is a donor and acceptor partner, it is possible tohave mived types; forexample, an electron-rich a-cloud610 / Journol of Chemiml Educotion


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can be a donor to a hydrogen which has the electron de- S + F X F;? S X + F

Although the bonding energies in kcal/mole of thesedonor-acceptor complexes have been measured in inertsolvents, these measurements represent maximal values,since the binding to enzymes occurs in water wherewater molecules can also form donor-acceptor complexes(4).The second class of complex formation involves by-drophobic bonding and the accompanying van derWaals forces; his isshown diagrammatically in Figure1,where the circles represent water. The solution of the

Figure 1. Hydrophobic bonding.hydrocarbon portions of A and B into water requires ell-ergy due to a change in water structure. When the solu-tions are mixed, a complex between A aud B moleculescan form with regeneration of th e origiual water struc-ture; thus part of the energy required to dissolveA andB in water will be released when co m~lexormation oc-curs (4).This process of hydrophobic binding is highly favor-

In order for the group X to be transferred to the sub-strat e, the group X must be held in juxtaposition be-tween F and S by the active site of the enzyme as shownin Figure 2; if more than a single interatomic distanceexists betwcen S and X, then new bond formation toform SX will be difficult. If the groupX is replaced byB (Fig.3 ), an inhibitor resultsifB cannot be transferred.If B is a leaving group that is juxtaposed to a nucleo-philic group within the active site, then covalent bondformation will occur which stops dissociation of the en-zyme-inhibitor complex, and the active site becomesselectively denatured; since this reactiou occurs insidethe active si te it is called active-site-directed irreversibleiuhibition by th e endo mechanism (1-3). This endomechanism has two serious drawbacks, namely (a) the Bgroup must be no larger than X or it cannot fit withinthe active site, and (b) the identical enzyme from differ-eut species or even mechanistically related enzymes willalso be iuactivated by FB.Exo Mechanism. If the leaving groupB (Fig. 4) is ona side of the inhibitor uot in contact with enzyme and isplaced so that it cau bridge back to some nucleophilicgroup on 1,he enzyme surface outside th e active site, thencovalent bond formation could take place (shown byarrow) (1-5). The irreversible inhibition by th e exomechanism overcomes the objections inherent in theendo mechanism since (a) large groups can be used be-tween A aud B, and ( b ) it is outside the active site whereevolutionary changes of amiuo acids can occur mostreadily-that still retain a function enzymeresu lt ing inthe possibility for differential irreversible inhibition ofthe identical euzyme from different tissues or species( 1 1 ) .

able for complex formation and has a maximum free en- Lactic Dehydrogenaseergy release of 0.7 kcal/mole per >CH,. . CH2< inter- Lactic dehydrogenase is an enzyme that oxidizes lac-action (8,Q). n addition there call be further affinity tate to pymvate mediated by DPN or reduces pymvateof A aud B due to van der Waals forces or dipole-dipoleinteraction; the van der Waals forces cau also have a to lactate with DPKH (eqn. (8)).maximum bonding energy of 0.7 kcal/moleof methylene-methylene interaction ( 8 , l O ) . Thus simple hydrocar- CH ~ C HCOO ~H ~ C C O O ~IOH IIbon bonding could have as much as 1.4 kcal/mole per x0 (8)methylene on the substrate or inhibitor, or about onepower of ten in the dissociation constant . When hydro-phobic bouding with an inhibitor can be fouud, it is a DPN DPNHmost useful phenomenon, as will be discussed later.Types of Active-Site-Directed IrreversibleEnzyme Inhibitors

Zndo Mechanism. Enzymatic reactions utiliziug acofactor can be written in the general form of eqn. (7).m, Figure 4. A simplified diagram of an ov e n i r e(noncla~siso l) inhibitor. Note that the excesssire above the horizontal dotted line of sub-strate faces away from the enzyme surface.Figure 2. A rimpliRed diag ram of the enzyme - Figure 3. A r impliRed diogram of endo- Wh en the aikylating group B can br idge to ocatalyzed transfer of the group X from o so- allylotion. A group, B, replaced the trow fer nvcleophilic rite on the enzyme surface adjacentfoctor F to the substrate 5, both molecules group X in Figure 1. B son then olkylote mm e to the octive $te , covalent bond formotionbeing jvxtoporitioned at the active site of the nucleophilic group within lend01 the active site lor row ) occur, outside the active site (em-enzyme. From refere nce( 21. to form o covalent bond. From reference (2). olkylotionl. From reference (21.Volume 44, Number 10, October 1967 / 611


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The main binding by lactate is through its caborxylateand hydroxyl groups. This binding can be simulatedby the o-hydroxyl and carboxylate groups of salicyclicacid; since the carbon of salicylate hearingthe hydroxylgroup does not also bear a hydrogen for transfer toDPN, salicylate is a reversible inhibitor. Salicylate canhe converted to an irreversible inhibitor of the exo-typeby placement of a function on the benzene ring tha t hasa leaving group (Fig. 5).

-Skeletal Moscle LDH- -Heart LDFI-K I Rate of K I Rate ofComod. 1mMi Inactivstion imMi Inaetmrsl~on

Figure 5. Selective irreversible inhibition of lactic dehydrogenasefrom different tisuer.

4-(1odoacetamido)salicylic acid ( I, Fig. 5) forms a re-versible complex with lactic dehydrogenase from eitherskeletal muscle or heart; however, only the skeletalmuscle enzyme is irreversibly inhibited by I (Fig. 5) witha half-life about 30 min (11, 13). When the distancebetween the carboxylate and the leaving group islengthened as in I1 (Fig. 5), then both enzymes are ir-reversibly inhibited; the heart enzyme is inactivatedwith a half-life of about 60 min (11, IS). When theleaving group is changed to pbenoxy, at the 4- or 5-posi-tion (Structures I11 and IV, respectively), both enzymesare reversibly inhibited about equally by both com-pounds; however, only I11 (Fig. 5) inactivates theskeletal muscle enzyme and only IV (Fig. 5) inactivatesthe heart enzyme-a crossover in specificity (11, 14).Since reversible inhibition still allows the enzyme to con-vert substrate to product, but irreversible inhibitiongives a non-functional enzyme, only the irreversible in-activation is important with an inhibitor of the type inStructures I-IV given in Figure 5.Dihydrofolic Reductase

Dihydrofolic reductase is an enzyme tha t can converteitherfolicacid I1 (aB-vitamin) or dihydrofolic acid I11to the cofactor form, tetrahydrofolate IV. There aretwo main classes of inhibitors of this enzyme (15, 16).

The first class are close analogs where the 4-0x0 group offolic acid, (II ), has been replaced by amino, as in V andVI ; both V and VI are extremely potent inhibitors of di-hydrofolic reductase and VI, known as methotrexate, isused for treatment of leukemia. The second class doesnot contain the p-aminobenzoyl-L-glutamate moiety offolic acid 11,but has instead a hydrocarbon like moiety;for example, VII is a good antimalarial agent known asDaraprim and VII I is a good antibacterial agent knownas trimethoprim. The hydrocarbon groups at Ra arecomplexed to a hydrophobic bonding region of dihy-drofolic reductase that is adjacent to the active site,but not part of the active site; evidence has been foundthat this hydrophobic bondiug region is adjacent t o thearea where the4-oxogroup of folic acid (11) resides on theenzyme (16-18); thus the pyrimidine of VII and VIII isshown with the R5 group in the area where the &ox0group is shown in 11,when they are complexed to the en-zyme.

Dihydrofolic Acid

Tetratiydrofolic Acid

V , R - HVI, R=CH,

&&HZN v NNH2

VII :RS p-ClCsHa; Rs=CaHsVIII: RS= ,4,5-(CHaO)aCsHGH2; Ra= H612 / Journal of Chemical Education


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Hydrophobic Bonding. Since this hydrophobic bond-ing region is not part of the active site, it can be an-ticipated that evolutionary changes would have oc-curred in this region; therefore one should expect toobserve differences n the ability of hydrocarbon groupsto bind to this region as enzymes from various speciesare examined. Some comparisons are shown inFigure 6 with the dihydrofolic reductases from pigeonliver, E. coliB, and the enzymeinduced by the bacterialvirus, T,phage, when it infects E. coli B.

pM Conc. for 50y0 InhibitionPigeon Tr E . eoliNo. R Liver Phage B

V Trimethoprim 16 0 . 6 8 0 . 0 0 0 3All assays with 6 rmM dihydrofolate.

Figwe 6. Species differences in hydrophobic bonding to dihydrofolicreductare b y the given structures.

The results in Figure 6 were selected from somefifty compounds that were assayed (19). Note thatthat phenyl binding (I in Fig. 6) with the Trphageenzyme is similar to pigeon liver enzyme but differentfrom E. coli B enzyme. Even more striking are theresults with the antibacterial agent, trimethoprim(V in Fig. 6), which has previously been shown to bemuch more effective on bacterial dihydrofolic re-ductases than the mammalian enzymes (20, 91).Note that structure V (Fig. 6) complexes 50,000-foldbetter to the dihydrofolic reductase from E. coli Bthan the enzyme from pigeon liver and 2300-foldbetter than to T2-phage iuduced enzyme. In contrastthere is only a 23-fold difference in binding to thepigeon liver enzyme and the Tz-phage nduced enzyme.However, there are some similarities in common withthe 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 liverenzyme, but only a 2-fold and an 8-fold loss, respec-tively, 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, buta 12-fold loss in binding to the pigeon liver enzyme.The results in Figure 6 give considerable insight

into the evolutionary differences in preferred con-formations of hydrocarbon groups for binding t othis hydrophobic region (16). The T,phage in-duced enzyme has some striking similarities to avertebrate enzyme such as that from pigeon liver;however, there are also similarities between the Tz-phage induced enzyme and the host bacterial enzyme.These results have some important hearing on the timein evolution when th e T,-phage emerged; pigeon andTrphage diverged from a common ancestor muchlater on the paleontological time scale than E. coliB, and pigeon diverged from a common ancestor.Such time sca'e divergences are more accurately doneby determination of linear sequences of amino acidsof the pure enzyme from different sources (22, 23);such studies with dihydrofolic reductase have the ad-ditional difficulty hat only 0.001-0.005% of the totalcellular protein consists of dihydrofolic reductase;it is therefore difficult to obtain sufficient amounts ofpure dihydrofolic reductase for amino acid sequencestudies.Hitchings, et al. (21) have been fabulously successfulin finding 2,4-diaminopyrimidines with varying hydro-carbon groups such as VII that are successful agentsfor treatment of bacterial and protozoan diseases;it is highly probable tha t the selectivity of action isdue to the 50,000-fold or more differences in bindingto the hydrophobic bonding region of dihydrofolicreductase where evolutionary changes had occurred.Such large differences would not be expected amongthe tissues of an animal. The largest differences wehave observed (19) in hydrophobic bonding to di-hydrofolic reductase from different mammalian tissueshas only been 100-fold; somewhat larger differencesmight be anticipated with a tumor inducing virus,but eveu these differences will probably not be largeenough to exploit with reversible inhibitors.Active-Site-Directed Irreversible Inhibition. It shouldbe possible to greatly magnify small differences in thehydrophobic bonding region of dihydrofolic reductases-from a tumor and normal tissues~bybringing in theextra parameter of active-site-directed irreversibleinhibition; such an approach is shown in Figure 7

(24). The maximum specificity should be observed

I 'Figure 7. Schematic representation of an active-sibdirected irrevers-ible inhibitor that utilizer hydrophobic bonding for specificity; AS =Active site binding of on inhibitor; HP = hydrophobic binding of oninhibitor; and Nu = an enzymic nucleophilic group.

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if an alkylating function on the inhibitor can he bridgedhack from the hydrophohic region on the enzyme to themore polar nucleophilic region. In this way the abilityof an irreversible inhibitor to bridge between thepyrimidine locus in the active site and the enzymicnucleophilic site can he controlled by the nature of thehydrophobic site, which is outside the active site andwhere the greatest evolutionary diierences can occur(11). It should also he possible to oht,ain specificityby varying the group that complexes in the hydro-phobic region; this could alter the position of thealkylatimg function (dot,ted arrow, Figure 7) by posi-tioning the pyrimidine slightly differently; this canbe likened to a fulcrum where a slight shift on thehydrophobic side will shift the alkylating side in theopposite direction where a part of the pyrimidine isthe axis of the fulcrum.

% %.Source K , X 106M uM Cone. E...I Inactivat,ionPigeon liver 0.09 0.25 80 0E . eoli 0 1 0 . 1 50 33

Incubated 2 hr with 30 uM T P N H a t pH 7.4 and 37" C.Figure 8. Selective inhibition of dihydrofolic reductass from differsntspecie. by the obove compound.

Demonstration of these predictions (24) soon fol-lowed. In Figure S is shown (25) selective irreversibleinactivation of B. coli B dihydrofolic reductase with-out inactivation of t he pigeon liver enzyme; note that,

reversible inhibition is the same with both enzymes.Compare the structure in Figure 8 with the diagramin Figure 7 where the hromoacetamido group is partof the dotted line. If the chloropheuyl group in Figure8 is changed to phenylbutyl, reversible binding to bothenzymes is still excellent, hut now neither enzyme isirreversibly inhihited-demonstrating the fulcrum prin-ciple above.Better irreversible inhibitors soon emerged (26)as shown in Figure 9 with the enzyme from mouseL-1210 leukemic cells. The compound in Figure9 could inactivate the leukemic enzyme with a half-life of about 5 miu in the presence of TPNH.Similarly, the enzyme from the Walker 256 tumorfrom the rat and the enzyme from rat liver could heinactivated with a half life of < 2 min. In contrast, theenzyme from pigeon liver was inactivated more slowly.In the absence of TPNH, the rat tumor enzyme wasinactivated with a half life of less than 30 sec. Syn-thesis of related compounds to build in more selec-tivity-by utilization of principles previously out-lined (2, 11)-into irreversible inhibitors related tothe compound in Figure 8 are underway; i t is withinthe realm of practicality to obtain compounds which,by the active-site-directed mechanism, will inactivatethe enzyme from a tumor such as the Walker 256with mineral irreversible inhibition of the enzyme fromnormal rat tissues.Hydrophobic Bonding to Other Enzymes

Enzymes that have been investigated for hydro-phobic bonding in this laboratory are listed in Figure10. If a hydrophobic honding region adjacent to theactive site can be found, t is a most useful phenomenon-as described in the previous section. In not allcases can a hydrophohic bonding region be found;for example, an extensive search on thymidine kinasefailed to reveal such a hydrophobic region (27, $8).No useful hydrophohic region has yet been found onthymidylate synthetase, but not all areas on theinhibitor have been investigated (29). Although weakhydrophobic honding to sucdnoadenylate kinosyn-

N@CI = He,.N H A ~K I X 106 fiM Conc. 0'o TimeC o m ~ o u n d M Inhibitor E . . . I (mi") %Inaet~vation

H~~


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I Piaeon liverR i t l iv erDihydrofolic Reductase Walker 25 6 mt tumurL-1210 .Mome LeukemiaE . coli BTrphage inducedThymidylrtte Synthetase E. coli BThymidine Kinase E. eoli BThymidine Phospharylase E. coliBGuanine Deaminase Rabbit liverXmthine Oxidase MilkSuccinoadenylate Kinosynthetase E. coliBFigure 10. Enzymes investigated for hydrophobic bonding.

thetase has been found with 8- and 9-substitutedadenines (SO), other areas on the adenine moleculehave not yet been explored. Hydrophobic bondingregions have been found on xanthine oxidase, guaninedeaminase, and thymidine phosphorylase; these arediscussed in the following sections.Thymidine Phosphorylase

This reversible enzyme (Fig. 11) can either converta pyrimidine deoxynucleoside to the pyrimidine orvice versa depending upon the stress of environment or.genetic deficiency. The main interest in our laboratory

+

+Phosphate OH

R=H, CH,, F, Br, CI, IFiguro 11 . Thymidine phorphorylare.

revolves around the ability of this enzyme to cleave theanticancer agent, 5-fluoro-2'-deoxyuridine, to 5-fluoro-uracil which is then further detoxified by other enzymes(28). If this enzyme could be blocked by an active-site-directed irreversible inhibitor in a tumor with-out blockade of the enzyme in normal tissues, a use-ful adjunct to treatment of tumors with 5-fluoro-2'-deoxyuridine (FUDR) could arise (28).Pyrimidine Binding. The mode of pyrimidinebinding is indicated in Figure 12; the 1- and 3-hydro-gensarecomplexedto the enzyme (as elect,ronacceptors),but the 2- and 4-0x0 groups are not (28, 81). Theability of the 1-hydrogen to bind t o the enzyme as anelectron acceptor is increased with increasing acidity;in fact, if tbe 1-hydrogen is sufficiently acidic so that

Bonded: @ , ($N ot Bonded: @ @

Figvre 12. Mode of uracil complexing to thymidine phorphorylose.

an anion is formed-as in the case of 5-nitrouracil(Fig. 13) even better binding is observed. Note that5-hromouracil,which s a considerably stronger add hanuracil, is complexed 9-fold better than uracil. In-creased acidity with substituents at the 6-positioncan also be achieved; in this case, comparisons should

acidic % R. or R.Rs R, I I S . pK . Ionired =-Constant~H H 3 . 9 9 . 5 0 . 0 3 OBr H 0 . 4 5 8 . 0 0 . 7 8 0 . 8 6N O z H 0 . 2 2 5 . 3 8 0 - 0 . 2 8H CHa 8 . 0 9 . 7 0 . 0 2 0 . 5 6H CF I 1 . 2 5 . 7 6 1 1 . 3II -S02CH3 0 . 5 5 ~ 5 . 4 ~6 - 1 . 3

Estimated from pK, 5.4 for 6dfam oyl-oraci l .Substrate: 0.4 mM FUDR. Source: E. coliB.lI/S)o.s = ratio of concentrations of inhibitor to substrate giving50% inhibitionFigure 13. Mode of uracil complexing to thymidine phorphorylose byvarious rub,tituted uracilr.

be made with 6-metbyluracil. 6-TriAuoromethyl and6-methylsulfonyluracil complex 7-fold and 14-foldbetter, respectively, to the enzyme than 6-methyluracil.Hydrophobic Bonding. Some uracils substituted byhydrocarbon groups at the 5- and 6-positions weresynthesized (81) in order to investigate hydrophobicbonding to thymidine phosphorylase; the results arelisted in Figure 14 (28, 81). Hydrophobic bonding

HCH sCsH,HHHHHH1%n-CsHn

Substrate: 0.4 mM FUDR. Source:

3 . 91.9-2

8 . )>5

0 . 2 20 .601 . 12 . 51 . 10 . 6 0

E. oli B.Figure 14. Hydrophobic bonding to thymidine phorphorylare with 5-and 6-substituted uracil%

could he detected with substituents at either the Ceor Cg position; the best hydrocarbon honding wasobtained with 6-benzyluracil which was complexed18-fold better than uracil.Substitution of a nitro group on the benzyl at eitherthe 3- or 4-position gave enhanced binding (31, 32)(Fig. 15); th at this result was not due solely to a minusHammet sigma effect was indicated by the failure ofthe strong electron-withdrawing sulfonamido group togive a similar effect; in fact, the 4-sulfonamido groupwas actually detrimental to binding (51).Since the 5-bromo group or uracil gave a %fold incre-ment in binding over uracil due to increased acidityVolume 44, Number 10, October 1967 / 615


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(Fig. 14) and the 6-benzyl group gave an l&fold incre-ment in binding over uracil due to hydrocarbon inter-action (Fig. 151, both groups were incorporated intothe uracil molecule; note that the two effects wereadditive (Fig. 15) and that 5-bromo-6-benzyluracil

Figure 15. Mode of phenyl binding to thymidine phorphorylose byvarious 6-benzyl urocilr.

is complexed to the enzyme 40-fold better than thesubstrate, 5-fluoro-2'-deoxyuridine (FUDR). In con-trast, introduction of a 5-bromo group on 6-(p-nitro-benzyl) uracil was actually detrimental to binding;the reasons for these contrary results are still obscureand are still being investigated.When a p-bromoacetamido group was placed on 6-benzyluracil, an active-site-directed irreversible in-hibitor of thymidine phosphorylase was obtained (52);in contrast, the m-bromoacetamido isomer was notan irreversible inhibitor, although it was reversiblycomplexed to the enzyme as well as the p-isomer.Further studies with related synthetics as candidateirreversible inhibitors are underway, with particularemphasis on species and tissue specificity.Guanase

Guanase (guanine deaminase) is a catabo1i.c enzymethat degrades guanine to xanthine. The mode ofbinding of the substrate, guanine, to guanase is in-dicated in Figure 16. The 1- and 9-hydrogens are

8Other Substrates:

complexed to the enzyme as electron acceptors andthe 6-0x0 and 7-nitrogen groups as electron donors(55, 54). Other substrates are the cytotoxic agents,thioguanine and 8-azaguanine, which are detoxifiedby this enzyme. The best inhibitor known prior toour studies (55) was 5-aminoimidazol&-carboxamide.Our objective with this enzyme is to find an active-site-directed irreversible inhibitor which mill blockguanase in tumor tissue with minimum blockage ofguanase from normal tissues; such a compound couldbe used for cancer treatment in conjunction with thio-guanine or Sazaguanine.

uM Cone.for 50%R Inhibition [I/SIs.a-CHs 275 21-CsNs 0 . 7 0 . 7 3-CHxCeH6 370 28-C.Hwn 450 34- ( C H M s H a 230 17

Substrate: 13.3 pM guanine. Source: rabbit liver (commer-cial).Figure 17. Hydrophobic bonding to guonare with voriour substitutedguonine,.

When the 9-hydrogen of guanine (Fig. 17) is re-placed by methyl, a 21-fold loss in binding occurs dueto the loss of this binding point. However, replace-ment. of the 9-methyl group with 9-phenyl gives a 29-fold increment in hydrocarbon binding. 9-Phenyl-guanine binds to guanase slightly better than the sub-strate, guanine; this replarement points up the im-portant concept that a binding point can be removedfrom an inhibitor without net loss of binding if bind-ing can be increased at some other part of the inhibitor.Note (Fig. 17) that hydrocarbon int,eraction withguanase is not effective with hydrocarbon groups otherthan phenyl.The mode of phenyl binding to guanase was theninvestigated (Fig. 18) (55). Note tha t the polar p-

UM Conc. fo rR 50% Inhibibion II/SIO.S

Inhibitor:

Figure 16. Guanasa (Guanine d e o m i n d . Figure 18. Mod e of phenyl binding to gumwe.616 / Journol of Chemicol Education


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carboxylate group, resulting from nearly full ioniza-tion of the carboxyl group a t pH 7.4 of the assay, isrepulsed from the enzyme. That this replusion is dueto lack of compatibility of the solvated carboxylategroup with a hydrophobic region on the enzyme, andnot due to a coulombic replusion of t he carboxylate byan anionic group, is indicated by the poor inhibitionof the p-dimethylamino compound; if poor bindingby the carboxylate were due to coulombic replusion,then the identically positioned dimethylamino groupshould have had a high affinity for this supposedenzymic cationic group. Since the electron-donating3-methoxy group, the electron-withdrawing cyanogroup, and the p-chloro group affect binding by only3-fold or less, it appears that little charge-transfereffect is involved in complexing of this phenyl moietyto guanase.The fact that the p-methoxy group (Fig. 18) givesa 25-fold increment in binding is interesting; it isunlikely to be due to the influence of the methoxygroup on benzene binding, else the m-methoxy, p-cyano, and p-dimethylamino should also have shownsome electronic effects on benzene binding. There-fore it is probable t ha t the p-methoxyl group is directlycomplexed with the enzyme, but further study will beneeded to elucidate the type of binding.Xanthine Oxidase

Xanthine oxidase is a catabolic enzyme that nor-mally converts hypoxanthine and xanthine to uricacid (Fig. 19) (36); 2-hydroxypurine and 8-hydroxy-purine are also substrates (37). In order for this

Other Substrates:

Figure 19 . Xonthine ox idare .enzyme to oxidize purine at any one of three positionson the ring, it is highly probable that the ring systemis not fixed in the active site, but any one of severalrotomers can complex in order to give optimum align-ment of the position on the purine to be oxidized withthe catalytic site (58). Xanthine oxidase can alsodetoxify the antileukemic agent, 6-mercaptopurine,(Fig. 19) by oxidizing it to the nontoxic thiouric acid(39); thus a tumor specific irreversible inhibitor of thisenzyme would be a useful adjunct to 6-mercaptopurinetherapy of cancer.That xanthine oxidase has bulk tolerance for largegroups in the 2- or 8-positions is indicated by the effec-tive inhibition of the benzylthiohypoxanthines inFigure 20. Although the bromoacetamido derivativein Figure 20 was a good reversible inhibitor of theenzyme, it failed to show irreversible inhibition; whenthis R2 group was placed on the 8-position (IX), re-versible inhibition was still good, but now (IX) could

Substrate: 8.1uM Hypoxanthine. Source: Milk (oommeroid).Figure 20. Inhibition of x a nt h in e o x i d a r e b y ~ b s t i t u t e d h y d r o x y -purines.

inactivate xanthine oxidase by the active-site-directedexo-mechanism with a half-life of about 50 min (40).

Hydrophobic bonding with 9-substituents on guaninewas then investigated (Fig. 21) (41). Guanine was an

PM Cone. forR 50% Inhibition [I/Slo.s

. .Substrate: 8.1 pM Hypoxanthine. Source: Milk (Commer-cial).

Figure 21. Inhibition of x a n th i n e o x i d o l e b y 9 - r u br t if u t ed g u a n i n e r

inhibitor of xanthine oxidase tha t was complexed5-fold less effectively than hypoxanthine, the sub-strate; a 9-methyl group on guanine gave little changein binding, indicating that the 9-hydrogen of guaninewas not needed for binding. Among other hydro-carbon substituents in Figure 21, only the phenyl groupgave an appreciable increment in binding; the hy-drocarbon interaction by the phenyl group gave a 100-fold increment in binding (41).The mode of this phenyl interaction with xanthineoxidase was then studied (56) (Fig. 22). There was nocorrelation of binding with electron-donating or elec-tron-withdrawing groups which had only 4-fold or lesseffects on binding. The highly polar carboxylate groupwas not repulsed, but gave a 3-fold increment in bind-ing; this result contrasts sharply with the effect of thisp-carhoxylate on the binding of 9-phenyl-guanine toguanase.

Volume 44, Number 10, October 1967 / 617


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pM Conc. for50% Inhibition 11lSla.~

Substrate: 8.1 pM Hypoxanthine. Source: Milk (commer-cial).Figure 22. M o d e of phenyl binding t o xonthine orid ore.

Comparative Hydrophobic BondingThe hydrophobic bonding to five enzymes is sum-marized in Figure 23 (41). Note that the type of

hydrocarbon giving maximum hydrophobic bondingvaries with t he enzyme and with the position of thehydrophobic group on the inhibitor; for example, thein-plane phenyl group gives maximum hydrocarbonbonding with guanase and xanthine oxidase, hut higherphenylalkyl groups are more effective on the otherenzymes. Also note tha t the benzyl group on the 6-position of uracil gives maximum hydrocarbon inter-action with thymidine phosphorylase, but that higherphenylalkyl groups are more effective on the 1- and5-positions of uracil. Although the 9-phenyl groupon guanine is the most effective for hydrocarbon in-teraction with guanase and xanthine oxidase, suhstitu-tion on the phenyl group has shown that the enzymicenvironment in the region of 9-phenyl bonding is notthe same with the two enzymes; for example a p-carboxylate on the 9-phenyl group aids hinding toxanthine oxidase, but is extremely detrimental tohinding to guanase (31).Enzyme-Inhibitor lntemctions and Student Research

All three levels of students have performed the re-search described in this article-undergraduates, gradu-ates, and post-doctorals. What does the researchstudent learn in this biologically oriented organicchemistry field? Primarily he develops skills as asynthetic organic chemist with all the modem in-struments the organic chemist now uses. Althoughno mention of synthetic methods for the many com-pounds necessary for these studies is made in thisarticle, it is clear tha t we search for biochemicalanswers by synthesis of appropriate compounds. Eachstudent learns how to do enzyme assays-which areroutine analytical assayeso that he can understandthe significance of the numbers obtained; he soondevelops a skill in interpretation of the enzyme data andin projecting his future research based on the enzymedata obtained. Most of the routine data is obtainedin our laboratory by technicians rather than by stu-dents, unless on rare occasion the student prefers todo his enzyme assays.

Although most of the work described in this articleinvolves heterocyclic chemistry-primarily becauseI believe these make excellent teaching prohlems-other fields of organic chemistry can be pursued byproper choice of the enzyme system to be investigated;it is obvious that the inhibitors will be somewhat re-lated to the substrate in structure and therefore thetype of synthetic chemistry to be investigated isprimarily dependent upon the structure of the suh-strate such as nucleosides, nucleotides, carbohydrates,amino acids, terpenes, steroids, lipids, and assortedheterocycles related to the B-vitamins.The student becomes quite expert in biochemistrywith particular emphasis on the chemistry of metabolicpathways. Organic chemistry today can be dividedinto: (a) new reactions; ( b ) synthesis of novel struc-tures such as natural products; (c) new physical prop-erties; or (d) new biological properties. It is thislatte r frontier where chemistry meets biology and newhybrid fields are emerging such as molecular biology,molecular pharmacology, and biochemical paleontol-ogy; even newer fields not yet formalized with a namewill continue to emerge in the future which will allowthe student to pursue useful and interesting problemsin hio-organic chemistry for many years to come.Literature Cited( 1 ) BAKER, . R., Cancer Chemotherapy Repor k, 4 , 1 ( 1 9 5 9 ).( 2 ) BAKER, . R . , J . Pharm. Sei ., 5 3 , 34 7 ( 1 9 6 4 ) .( 3 ) BAKER, . R. , "Design of ActiveSiteDirected IrreversibleEnzyme Inhibitors. The Organic Chemistry of the En-zvmic Active-Site," John Wilev & Sons Inc.. New York,1967.(41 See reference (8). chanter 11.. , ,, .( 5 ) KOSOWER, . M., "Molecular Biochemistry," McGrawHill Book Co., New York, 1962, pp. 185-195.( 6 ) ANDREWS, . J., AND KEEPER,R. M., "Molecular Com-plexes in Organic Chemistry," Holden-Day, San Fran-cisco, 1964.( 7 ) BAKER, . W., AND SHULGIN,. T., . Am . Chem. Soe., 8 0 ,

5 3 5 8 ( 1 9 5 8 ) .(8) BELLEAU. .. AND LACASSE. .. J . M e d . Chem.. 7 . 7 68. . . . . . .( 1 9 6 4 ) .( 9 ) NEMETAY, ., AND SCHERAUA,. A,, J . Phys. Chem., 6 6 ,1173 (1962).-..-,( 1 0 ) SALEM, ., Can. J . Biochem. Physiol., 4 0 , 1 2 8 7 ( 1 9 6 2 ) .( 1 1 ) See reference ( S ) , chapter IX.( 1 2 ) BAKER, . R ., LEE,W. W., AND TONG, ., J . Theowt. Biol.,3 , 4 5 9 ( 1 9 6 2 ).( 1 3 ) BAKER, . R. , Bioehem. Pharmaeol., 11, 1155 (1962).( 1 4 ) BAKER. . R .. AND P.~TEL.. P.. J . Pharm. Sei . . 5 3 . 7 1 4. .( 1 5 ) For a review, see JUKES,T. H., AND BROQUIST,. P., in"Meta,hbolio Inhibitors," (Editors: HOCHSTER,.M., ANDQuastel, J. H. ) ,Academic Press, N. Y., 1963, pp.481-534.( 1 6 ) See reference (S), chapter X.( 1 7 ) BAKER, . R., SCHWAN,. J., NOVOTNY,., Ho, B.-T.,J . P h a ~ m . c i . , 55 , 295 (196 6) .( 1 8 ) BAKER, . R.,4ND sFI.4~1~0,. S., J . P h a rm. SCi., 55 . 308( 1 9 6 6 ) .( 1 9 ) BAKER, . R. , J. Med. Chem., 10 , Sept. ( 1 9 6 7 ) .( 2 0 ) BURCHALL,. J., .4ND I


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R for Maximum BindingEnzyme Source Heterocycle Hydrophobic Bonding Increment Reference

Uihydrofolic Pigeon LiverReductase

CH3

Thymidine E. coli BPhosphorylase

Xanthine Oxidase Ravine Milk

a Included for comparison.Higher phenyldkyl analogs not yet investigated, but lower andogs were less effective.Figure 23. Hydrophobic bonding to some selected enzymes b y heterocycle= with hydrocarbon wbstituents,

(26) BAKER,B.R., HUANG, . C., AND A. POGOLOTTI,. Med.Chrm., ubmitted.(27) BAKER, B. R. , 4ND SCRWAN,. ., J. Med. Chem., 9 , 73IlQR6>-"--,.(28) See reference (5), chapter IT.(29) See reference (3), chapter XI.

(30) BAKER,B. R., AND ERICKSON,. H., J . PhaVn. ad., 56, inpress (1967).(31) BAKER, . R., AND KAWAZU,., J . Med. Chem., 10, 31111967).,(32) BAKER, . R., AN D KAWAZU,., J . P k a m . S c i. , 56, in press119fi7>.

(33) ~ e i i e f e k n e e3), chapters V and VII.(34) BAKER, . R., J . Med. Chem., 10, 69 (1967).

(35) BAKER,B. R., AND WOOD,W. F., lo , Nov. (1967).(36) KALCKAR,. M., J . Biol. Chem., 167, 429 (1947).(37) BERGMANN,., LEYIN,G., KWIETNY-GOURIN,., ANDUNGAR, ., Biochim. Biophys. Acla, 47, 1 (1961).(38) BAKER, . R., AND HENDRICKSON,.L., J . Pharm. S&., 56,in press (1967).(39) ELION,G. B., BIEBER, ., AND ~ ~ I T C H I N G S ,.H., Ann. N . Y .A d . &., 60, 297 (1954).(40) BAKER, . R., AND KOZMA,.,J.Med.Chern., 10,682(1967).(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 . S ei., 54,1415 (1965).(43) BAKER,B. R., Ho, B.-T., AND LOURENS, . J., J. Pharm.Sei., 56, 737 (1967).

interaccion de enz. - inh

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