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Published by: Smeetha Kaur on Aug 27, 2011
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11/02/2013

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ARCHIVES

OF

BIOCHEMISTRY

.4ND

BIOPHYSICS

96, 28-31 (1962)

The

Integrated

Michaelis-Menten
F . A . HOMMES’

Equation

-1 critical consideration of the assumptions involved in the steady-state treatment of enqme kinetics showed that these assumptions are only partially correct. A new theory is developed, from which it follows that an eqnation of the type of the integrated Michaelis-Menten equation can describe an enzyme-catalyzed reaction without assuming a steady state, This implies that such a reaction is determined I))? all reaction constants involved, which can be determined by analog computation. INTRODUCTIOiS The rnt)c c*oust!ants of au enzyme-cat#alyzed reaction according to t’he equation:
F,+s kl -ES-E+X , x.3

the following moment:
n’s

equations

must, hold

at every

=
clt

-ICl(fj

-

p)S

+

li*p

“2

can be determined by measuring the rate of the reaction in the phase of tht prestcady state. A very specialized apparatus is required for these measurements (stop-flow technique) . In many casts only approximat,e values arc obtained owing t,o a lack of an adcquatc solution of t)hc mathemat,ical problrm (I, 2). The Jlichaclis-Mcnten constant, K,,, = (/iz + lia)/kl and k, can br obtained from Linewcaver-Burk plots (3). Howe\:cr t.his procedure gives no information about the ubsolut,e Values of /cl and 1~~. The object of this paper is to cxnmiue as to ho!v far values of lC1 ) /is , and /ia can be obtained Ivithout measuring the rate of w&ion in the prestrady state. THEORY If s = concentration e = total enzyme p = concentration complex ES r = concentration of substrate S concentration of enzyme substrate of product’ X

dP = Xl(f’ dt

p)S -

(h.2 +

Ii3)p

(2)

By a straightforward analysis it, can bc easily shown, t,hat, these three simultaneous differential cqunt’ions yield the rquat,ion :

relating This tion:

product equation

.I’ and time t. is identical wit,h

t,he equa-

1 Present address: Johnson Research Foundation, Universit,y of Pennsylvania, Philadelphia, Pa. 28

but this is not cxprcsscd by Eq. the relations so = s + p + J and 0 = (ds/dt) + (dp/dt) + (d. The order of the equat)ion can thus be lowered by the substitution: d!! -=q da q k” + (A . Hcrc.?‘ = 0. the deviation from t. This means that the assumptions made for the derivation of F:cl.ration is sufficiently high. of course. (8) will be only negligibly small if the substrate conccnt. (7) holds only at one moment. (8) arc not rnlid for this case. the substrat. If the substrate concentration is sufficiently high. (8).. i. we may put C(j) Combination of Xqs. The latt. (8) gives often an adrquatr description of an cnzymc-catulyzcd reaction. the reaction is 110 longer of zero o&r. 111 sois to the right-hand side of El. (7) gives: d2y dr2 + (d . this steady-state conditiou will last.r/dt = k:(c: . In that. As dpjdt must be small relatirc to ds/dt and da/dt. Howcvtr.E = 0 In principle.Tdoes not appear. Thr contjribution of the term K.:~ Twsus 1/s. . this cannot be solved in this cast because one has to solve cquat.r /‘dt3 can be dropped. but not so small t.‘dt* and tl”. namely at the moment.. (1) with cl”s. Eq. dp:dt is also small relative to c/s /‘rlt and tl.er is of the type: Integration of Eq. for every change in 1’ = d. In fact it is possible to measure dp/dt. Assuming a relation of the type 1 =i+r.ions of the type: /j = ax + hex.hermore.IYTEGRATED ILIICHAELIS-MESTEN EQUATIOS “9 which was derived by Swoboda (4).By)q dq Cq’ + Dy .ting (1 = l. (3). Nevertheless. So the steady-stat’e assumption can only giw zero-order reactions. as at t.. This is the formulation of a zero-order reaction. case a zero-order rractiou will be obtained.r/dt is reduced to a change in p.r/&) must. whew so is the concentration of substrate S at time t = 0.(2) gives a relation for (5) Furt. *%ii analytical solution is not possible. Thus: By stt.~.r = li&.. howwr. in other words.I3 = 0 In this equation . of the true steady state. for a reasonably long time. (b) d. (5).he strady state will be so small that it can be neglectrd.r/dt = 0: s is constant.By) 2 - C ’ + I)!/ .e concentration is of the same mng~litude as Ii.ion arc: (a) d. = 0. which can be solved in some instances only.c. than all ttrms in Eq. and thus dp/dt # 0. or is so small that any variation in p can be put equal t)o zero.Km ___ The two solutions of this equat. In these cases a straight line is obtained by plotting 1. hold. when u and s are detcrminrd at one s-t curve. However.! 1 2’ S . the system can be simplified by assuming a steady state for the intermediate cnzymc-substrate complcs: dp/dt = 0. Eq. WC may ask if a steady state is a necessary condition for this phcnomcnon.hat it can be ncglccted. or. . deviations from the steady state lwomc npprcciablt. This is only the case if no reaction occurs at all. Substitution of this condition in 11:(1.. dt. and (6) yields ds -2 k~es = s i. If dp/‘dt = 0. If.!u this can be transformed t>o: According to Kamke (5) this diffrrential equation belongs to the type given by Abel.

the11 ‘c:jc = kl!K. Ii2 .e. From the kinetics of this phase. this can be underst. (11) gives X.:jptso + K.L . so at that moment w-emay put s = so. (1) p cm be solved: 2 + X1es p = k. .ion (6). If t. can be determined by t. (9) as (s. .hat as t approaches infinity.ion of t.I‘ = X’:~cs.L)K.s) ( 13) Assuming a skady state. .K.s + I?2 (10) dP -= dt . = 0 1 + (s (J Substitution of Ii:q.he sub&ate concentration is sufficiently high.“. + Ii:{) “~1then Eo. (12) in 15~1.p)s = pK. 0 = /il(f’ poho - (/i:! + X. + . (3) yields: p = poePat. (10) in Eq.hc enzyme-subst. the presteady-st. 90 A~ssuming0 = (/ilk’ k'.j’ !I 1s ln ?’ Substitution of 2: = kap in Eq.as far as /I is really a constant. and X3can be determined.~/ + K.ood as a differential ecluation in s and t.ip ---~ dt The concentrat. This can be written as: 1 ds 1.K) si.hc relation 01 = I<:$(. the st)eady state .atc values of kl ..) t + ‘~:.he considerat8ion that at. (8).)] [cx(. . K = K.K) Substitution of Eq. (9’) yield:: (c ..rate complex rises wry rapidly in the initial phase of an enzyme-camlyzcd react.& ‘i.s” (1 ..l/l.. which can be solved: ( ‘l. i. relative to K. This maximum is reached in the ycry early phase of the rcaction. K..)(s. p..:j)p. gives t... We may write Ey.)..30 HOMMES From Ey. Equation (2) can now be written as follows: dP = /<I(K ./ (so + K.. + K.‘dt = 0. t.Cmust approach s0./iae. (11) so.‘~ is identified with 1 :/c~(J:n = /i:ie and 1 ds Integration yields .aP1 with a = kr(k.. = (I<. Substitution of the boundary condition t. + Km - ‘(2) t + : 111 sI SC. + c ) ( = (SC.he maximum of an experimental p-t curve indeed dp..b 111 s + y s (’ = c Hy definition of N: f ’ = ‘cab 111 SIJ + .eat). Thus Insofar as 0 is really a wnstant. (11) reduces to ]‘:(I.

i:~lgleichu[lgerl. 6.r-t curve is completely idcnt icul with an ex- perimentally determined .s (‘an be found that a simulated .. 1. 63. 01dy. 2. 656 (1056). 5. In such cases t)hcserate parameters can be perhaps determined by analog computer experiments in which by trial and error such values of I..ype of the integrat. (. “nifi’crent. . s. .r-t curve. H.\‘o. T’erlagsges. 151. and 1. H. If it is pcrmissiblr that. then this time course is not predicted by k3 and K. is obvious that the above-outlined t. . I. .will he maintained during a cert.. It. . unt. J.ain time.CTFREI~NU. .. this point’ deviat’ions from the steady state become appreciable. liz . Sri..heory is only valid if s. M. 719 (1956). /lid. KAMKE. 553 (1943). :~cnrl. J. An equation of the t. l’reliminary experiments with the syst’em chloroacetyl-L-phenylalunine-carboxypeptidase . 1943. lvithout assuming a steady stntc of the intermediate enxymcs&skate complex.Yatl. hut by /iI . 220 (1954). 1. . I..” Ak:d. C~rasm. the complete time course of such a system can be described wit. nwthoden uncl Lbsungen. 42. aild I<3. Proc~. is of the same magnitude as K. R. 24..ijsungsVol. ASI) STI-RTEVANT. 17. (‘hem.. At.il the substrate concentration becomes of the same magnit#ude as K.hout assuming a steady stat’e. Rio&u/...A have shown that this is indeed the case. I. C.ed Michaelis-Mentcn equation can describe such a system. F:. Discussions Fat~aday SW. Leipzig. J. GL-TFRECNI). It implies an exponential breakdown of this in the prcstcndy-state phase of the react ion to a maximum value in creasing complex.

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