You are on page 1of 4






96, 28-31 (1962)





-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 the equation:
F,+s kl -ES-E+X , x.3

the following moment:



must, hold

at every





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 +



By a straightforward analysis it, can bc easily shown, t,hat, these three simultaneous differential cquntions 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




which was derived by Swoboda (4), whew so is the concentration of substrate S at time t = 0. The latt,er is of the type:


of Eq. (7) gives:


dr2 + (d - By) 2 -

+ I)!/ - I3 = 0

In this equation .Tdoes not appear. The order of the equat)ion can thus be lowered by the substitution:

d!! -=q da
q k + (A - By)q dq Cq + Dy - E = 0

In principle, Eq. (7) holds only at one moment, namely at the moment, of the true steady state. If the substrate concentration is sufficiently high, this steady-state conditiou will last, for a reasonably long time; or, in other words, the deviation from t,he strady state will be so small that it can be neglectrd. In that, case a zero-order rractiou will be obtained. If dp/dt = 0, or is so small that any variation in p can be put equal t)o zero, than all ttrms in Eq. (1) with cls,dt* and tl.r /dt3 can be dropped. Thus:

By stt,ting (1 = l,!u this can be transformed t>o:

According to Kamke (5) this diffrrential equation belongs to the type given by Abel, which can be solved in some instances only. Howcvtr, this cannot be solved in this cast because one has to solve cquat,ions of the type: /j = ax + hex. *%ii analytical solution is not possible. However, the system can be simplified by assuming a steady state for the intermediate cnzymc-substrate complcs: dp/dt = 0. Substitution of this condition in 11:(1.(2) gives a relation for

Furt,hermore, the relations so = s + p + J and 0 = (ds/dt) + (dp/dt) + (d.r/&) must, hold. As dpjdt must be small relatirc to ds/dt and da/dt, we may put

Combination of Xqs. (3), (5), and (6) yields ds -2 k~es = s i- Km ___

The two solutions of this equat,ion arc: (a) d.r/dt = 0: s is constant. This is only the case if no reaction occurs at all. (b) d.r/dt = k:(c: .r = li&, as at t, = 0; .? = 0. This is the formulation of a zero-order reaction. So the steady-state assumption can only giw zero-order reactions, but this is not cxprcsscd by Eq. (8). Thr contjribution of the term K,,, 111 sois to the right-hand side of El. (8) will be only negligibly small if the substrate conccnt,ration is sufficiently high. If, howwr, the substrat,e concentration is of the same mng~litude as Ii,,, , the reaction is 110 longer of zero o&r; i.c., deviations from the steady state lwomc npprcciablt. This means that the assumptions made for the derivation of F:cl. (8) arc not rnlid for this case. Hcrc, of course, dp:dt is also small relative to c/s /rlt and tl.~; dt, but not so small t.hat it can be ncglccted. In fact it is possible to measure dp/dt, for every change in 1 = d.r/dt is reduced to a change in p, and thus dp/dt # 0. Nevertheless, Eq. (8) gives often an adrquatr description of an cnzymc-catulyzcd reaction. In these cases a straight line is obtained by plotting 1,:~ Twsus 1/s, when u and s are detcrminrd at one s-t curve. WC may ask if a steady state is a necessary condition for this phcnomcnon. Assuming a relation of the type 1 =i+r,! 1 2



From Ey. (1) p cm be solved: 2 + X1es p = k,s + I?2 (10)

dP -=

- aP1

with a = kr(k,, - K)

Substitution of Eq. (10) in Eq. (3) yields:

p = poePat,as far as /I is really a constant. p. can be determined by t,he considerat8ion that at, t.he maximum of an experimental p-t curve indeed dp,dt = 0. This maximum is reached in the ycry early phase of the rcaction, so at that moment w-emay put s = so. 0
= /il(f poho -



This can be written as: 1 ds

1,~ is identified with 1 :/c~(J:n = /i:ie and 1 ds Integration yields .I = X:~cs,~/ + K,)] [cx(.s (1 - eat). Substitution of the boundary condition t,hat as t approaches infinity, .Cmust approach s0, gives t,hc relation 01 = I<:$(,/ (so + K,,). Thus

Insofar as 0 is really a wnstant, this can be underst,ood as a differential ecluation in s and t, which can be solved:
( l.l/l.& i,) t + ~:,b 111 s + y s ( = c

Hy definition of N:
f = cab 111 SIJ + ; 90

A~ssuming0 =



c:jc = kl!K,,, - K) si, + Km


111 sI SC,

(11) so, = 0

1 + (s (J

Substitution of Ii:q. (12) in 15~1. (11) gives X.:jptso + K,, + c ) ( = (SC, K,)(s,, + - s)
( 13)

Assuming a skady state, i.e. K = K, = (I<, + Ii:{) ~1then Eo. (11) reduces to ]:(I. (8). We may write Ey. (9) as

(s,, + K,,L)K, - ;j !I

ln ?

Substitution of 2: = kap in Eq. (9) yield:: (c - p)s = pK. Equation (2) can now be written as follows: dP = /<I(K - K,,ip ---~

The concentrat,ion of t,hc enzyme-subst,rate complex rises wry rapidly in the initial phase of an enzyme-camlyzcd react,ion (6). From the kinetics of this phase, the presteady-st,atc values of kl , Ii2 , and X3can be determined. If t,he sub&ate concentration is sufficiently high, relative to K,,L , the st)eady state

will he maintained during a cert,ain time, unt,il the substrate concentration becomes of the same magnit#ude as K, . At, this point deviations from the steady state become appreciable. An equation of the t,ype of the integrat,ed Michaelis-Mentcn equation can describe such a system, lvithout assuming a steady stntc of the intermediate enxymcs&skate complex. It implies an exponential breakdown of this in the prcstcndy-state phase of the react ion to a maximum value in creasing complex. It, is obvious that the above-outlined t,heory is only valid if s,, is of the same magnitude as K,,, . If it is pcrmissiblr that, the complete time course of such a system can be described wit,hout assuming a steady state, then this time course is not predicted by k3 and K, 01dy, hut by /iI , liz , aild I<3. In such cases t)hcserate parameters can be perhaps determined by analog computer experiments in which by trial and error such values of I;, , I; , and 1;s (an be found that a simulated .r-t curve is completely idcnt icul with an ex-

perimentally determined .r-t curve. lreliminary experiments with the system chloroacetyl-L-phenylalunine-carboxypeptidase ,A have shown that this is indeed the case.



H., ASI) STI-RTEVANT, J. M., Proc~. .Yatl. :~cnrl. Sri. C. s. 42, 719 (1956); Rio&u/. J. 63, 656 (1056). GL-TFRECNI), H., Discussions Fat~aday SW. .\o. 17, 220 (1954).



F:., nificrent,i:~lgleichu[lgerl. I,ijsungsVol. I, 1,. 24. 1943. 151, 553 (1943).

nwthoden uncl Lbsungen, Ak:d. Terlagsges, Leipzig, R., J. /lid. (hem. 6. C~rasm,