6S6
Enzyme Inhibitors.
XVII. Kinetic Studies on the Irreversible Inhibition of Adenosine Deaminase
A kitietic method h:ih beeii developed for t,he evaluation uf the reversible etiz~ino~-iiihil)itt,l. tiinsttciatioii citit>stiiiiI of an enzyme by ati active-site-directed irreversible inhibitor. This method should be applicable to all irreversible enzyme inhibitors that, proceed t,hrorigh an initial reversible enzyme-inhibitor complex. It has been shown that irreversible inactivat,ion of adenosine deaminase caused by 9-(p-bromoacetamidobenzy1)adenirie (1) and by '3-(o-bromoac'etamidobeiizyl)adenine(3) is ttot a raildom bimolecular procesrj but proceeds by a first,-order react,ion in an initial reversible enzyme-inhibitor complex. ITsing this new method, the K i of 1 and 3 were found t o be 1.4 X 10-j .TIand 43 X 10-5 M , respectively, in cscellerit agreement with the values obtained previously by t he rtaual doiible reciprocal plot rnet,hod. Furthermore, i t was forirrd that, 3 alkylates the enzyme about aeveit time.? more rapidly than does 1. The first-order ratr roltrt,aiit ( k 2 )for the alkylation iii the reversible E-I complex for 1 = 1.1 X niiiir' and for 3 = 7 . 7 X IO-'
(Ki)and of t8hefirst-order rate constant for the alkylation
1iiiii-I.
During the past several years, a number of lahorxtorics have been interested i l l the design arid syrithe of irreversible inhibitors of certain enzymes.2-4 studies on esterases arid irreversible inhibitors, equatioris have been derived which allow one to evaluate K , (the affinity constant) and k , (the phosphorylation rate const'ant) or kzc (the carbamoylation rate contnt of the enzyme).j In this paper we wish to present M new mat,hematic*aldescription of the irreversible itihibitioii o f an enzyme which allows une to det'erminc Ki and k , (the first-order rat,e cwnstarit for the alkyhtioti of t.he enzyme by the inhibitor). Several recent papers have desrribed our research o i l the irreversible inactivatiori of calf intestinal mucosi adenosine deaminase by 9-(p-, ut- arid o-bromoaretILniidobetizyl)adeiiines.6 It was found that' the para and oi'tho derivatives (1 and 3) were much more effective itwversihlc iiihibitors of adenosine deaminase thaii \vas the ~ c t isomer a (2). I t tvas also possible to mewiii'c the itiit'ial revcrsihlc inhihitioil of adenosine deami1 1 : ~by ~ these compounds, and it n-vas found that the effectiveness of reversible inhibition decreases iti the followitig order: para isomer (1) > Illeta istrnier (2) > ovthuj isomer (3). Consequently the potency of thew ronipouiids its irreversiblc inhihitors i;i riot merely :I rcflcc*tiotiof their ability to form :t reversible romplcx r wliicd must' tx coilsidered ivheri conic inhibitors of enzymes is the chemiral reactivity of the alkylating o r acylating group of the itihibitor. -l-(p-l\jitrohe~izyI)pyridiiie ha.: been used previously to vompare the chemic*al reactivity of alkylatit~g Using 4-(p-tiit'roberizyl)pyridirle ive csonipared the reaction rates of iodoacetamide with 1, 2, uric1 3. If the rate with iodotretnmide was normal!11 This investigation \'ab supported by G r a n t T-337A from the American ('ancer Society, by a Public Health Service research grant (5-ROl-GM09775-051, by a research career program award (5-K3-CA-lST18-05) from the National Cancer Institute, and a training grant (5-T1-GM-555-05) from the Division of Medical Sciences, U. S. Public Health Services, Betheada, hld. (2) 13. It. Baker, J . I'hurm. Sci., 53, 347 11964). (31 G. Sclioelmann and E. Sha\v, Biochemistry, 2 , 252 (1963). Gchramm, ibid., 4 , 377 (1965). ( 5 ) (a) .I. R. Main, Science, 144, 992 (1961); (b) .4. R. hlain a n d E'. Iverson, Biochem. ,l,,100, 625 (1966): i c ) h. R. 11ain and I'. I,. Hasting-. & C l F n C E . 164, -100 (1066). (6) (it) 11. ,J. Schaeffer arid E. Odin. .1. -IIet/. C h e m . , 9 , 376 ilLl136~; ( 0 ) I f . ScliaeBer and R. N. .Johnson, J . I'hurm. Sci., 66, 929 (1966); ( c i 11. .J. IiaefTer and E. Odin. J . X e d . Cliem., 10, 181 (1987).
2"
NHL I
I
I
NHCOCH,Br NH2COCH2Br
2
1 "2
ized to 1, theti the rnte5 with 1, 2, a i d 3 n e i ~;3.0, 1.1. and 5.6, re\pertively. Since this ordei, of mtc*ti\4ty does not reprewit the effectivetie+ of 1, 2, a t i d 3 :i\ irreversible inhibitori of :idenositir deitminaic.. i t I \ :qiparcnt that ti morc hubtle effert or :L wnititi:ttioii of wveral efferti determine the effeetivcric~*of :ti1 11'rever4ble itihibitor of the erizyrric. 111 older t o gatit inore iiizight into the mecharii~mof thii re:wtion. :I kinetic* itudy i d the irrcverhihle inactivxtioti ( i f :iclciiosine deaminase by 1, 2, and 3 was undert akcn. When ail enzyme i z irrever.ibly iiihibitetl by , t i i alkylating or acylatitig agent, it would appear that t he two moqt probable mechanisms for the iri:wtiv:itioti are (A) a bimcrlec3ular attack of the inhibitor o i l Ihc enzyme or (B) the initial formation of a reversible (.omplex through I\ hich cwvalent bond formation ( J W U Y ~ . These two niech:mi-m\ may be distinguished liiiirt icdly qince. it1 the ( e of the bimoledar mechnriim 17) la) ,J. Eiistein, 11. \ \ . Ru~eiitlial,and Iivation of adenosine deaminase, but the rates were t'oo low to allow an accurate kinetic evaluation due to considerable t'hermal inactivation of the enzyme over a 2448-hr period. I n the case of 1 and 3, the irreversible inactivation of adenosine deaminase was relatively rapid; therefore, the 1;hermal inactivatjon of %he enzyme was ignored. The apparent first-order rate constants for the irreversible inhibition of adenosine deaminase were calculated from the slopes of the lines shown in Figures 1 and 2 and a complete (10) (a) M. A. Schmarta, P h . D . Thesis, University of Wisconsin. 1959; (b) T. Higuchi. Progress Report submitted to Armed Forces Chemical Center, Edgewood, Md. (May 20, 1959); (e) T . C. Bruice and S. J. Benkovic, "Bioorganic Mechanism," W.A . Benjamin, Inc., New York, N . Y . , 1966, p 143. (11) B. R. Baker, Biochem. Pharrnacol., 11, 1165 (1962).
688
H. J. SCHAEFFER, 11. A. SCHWARTZ, AND E.
1-01. 10
ODIs
V IO
20
30
40
n 50
60
70
80
-
90
I
(I)
Figure 3--I'lot of l/k,,,,,l for irreversible iiihibitioti of :&tic)deamiiiase cs. 1/ [I] ( m n l ) : 0, !~-(p-bromoac.eta~nidol,er~~~.I)adenine (1): 1.!)-(o-bromoacetarnidober~z~l)ade~~i~le 13). rille
case of the oltho derivative (3), the A p e of thcl liiic 3.Z7 and the intercept i4 13. Therefore, K , = 4 4 X 10-5 J / arid k , = 7.7 x 10-2 niiii-I. We liavcl proviously determined that both 1 arid 3 are (*ompetitivc inhibitors of adeno4ne deaminase by the Liiicn c a v c ~ Burl< method arid found that the K , of 1 = 1.3 X 10V' A l l a i d the I
