V~l~nae 32, ~a~rnbe:r ] FEBN LE]q"~RN Blay 1973

A N A L Y S I S 'OF E X C H A N G E R A T E S F O R T H E IPlNG PONG BI B1 M E C H A N I S M

AND T H E CONCEPT O F S U B S T R A T E S Y N E R G I S M -

James D. LUECK and Hezbert J: F~OMM

D~'p~rt_,._nem o f Bioehem~str..~" ~nd Biophysics, 7o~a Sta~e Uni~,e~sity, Ames. 2~w~ 5~919~O. USA

Received 5 Marela 1973

I. InI~'ofl~elion k~ g:3
E+A~----EA. .'E P
B5~e~etmat enzyme mechanisms may be divided
imO two classe~, ~quenfiN mad Ping Pong []] and 1hey k5 ~7
may be r:~adily separated by ini~,~a]zme experiments E'+B. "EBb--2 E +Q
12]. The cardinal feature of ~he Ping Pong mechanism
5s ~hal ,the enzyane leacts wflh the s~bsI,mte to f~rm a
ploflucl which d~ssoeia'tes betbre ~he second substrale Scheme 1.
adds to the active site of the enzyme. In the case of
the sequential mechanism bmh subst~ates mu~t be
present on.lhe enzyme s'amub:aneously before product - % + + (1)
formation may ocear. A B
'One characteristic of the Ping Pong mechanism is
the requhemem for parda] e?~,changeteatlions; how.
ever, 'these exchanges are ~ften slow relative to the cDncem~afio~, initial ~e]oci'ly k3 - 7 /'(k ~kk.,].,
"
initial ~eacfion ~eaocity. Bridget e, M. [3] have (k2 +ks)/k,~ ~3, (]':6+k7)~'t:~k 7, ~ubztrme A and sub-
~1tempted to aceoum fo~ ~ese N o w partiN ,exchange mral B, r~spe~five]y.
~eaclion~ in Ping Pong me.ehanism~ by ~uggev ~g that Some iath~I tloublesome p~oblem~ have becor~e
if net reaction ~a,~e~are mp~d compared to partial apparem fin reeen'tyears in 'the case of Fang Po~g
rea.ctions, zyne:gism wo.u]d appea~ prob~b]e..We w i ~ rnechani~ns, One of these is ma~ s:qu~ntial mechan-
to s~aggest here ~hat before the concel~t of s~ibstrale isms may appem t.o be Ping Pon~ if the 4h 2/(A)(B)
synergism is Nv~ked an im_portam .expresNon wNch term is ~rn~l relative t.o .othe~ trams in eq- 1. Indeed,
re]aIes 'th~ partial exchange a.aI.esand irfi~tia]~elocifie~
must be zati~fied. brain h:exoldn~ase I5], phosphofmc~okiriase |6], and
D-amino acid oxida~e [7]. Tha~ the Ping Pong mechan-.
ism foI these ,enzyme~ an~y be ~m artifac,~ was recognize(
2. Theory .and disenssion using alt,ernati~r.esn ~si~a~.
One would expeet.,that an enzyme which exhibits.
,Scheme ] illustrates :he well knovzn Ping-BongBi " a Ping l:'on~gmechanismsho.'u]deatalyz~ So-e~ed h.~lf

" ,: .. ~- - .-. " i .-.i.:....." .::.. ":-..:i.:---/--. con~J-:,iom:;(a) The..enzyme pZep~aZion eoma~s:eon:
~rolume 32, .~urnbm ] FEBS LETTERS May t973

taminating activities which are ~espun~ible fox the (k2~6), where Vj-and ~ zepresem ~ e maxmaa~
paltial exchange(s) and ~he mechar~;sm i~ not ready bridal ra~es fo~ lhe f o ~ d a~-d rever~ z~a~ions,
Ping Pong; (b) EitheI ~he ,enzyme o i a par~.~culal sub- respectively.
strate--pmflucI p~fi~~s contaminmed by one ol both tn considering the fo~.-wmd~eacfio~ and th~ maxima]
of the substra.Ies and p~od~e~s; (e) The Phng Pong path- A ~-~ P ~xchang.e,ff k 2 >~ k 3 and k 7 >> k 3, ~ =
way is no~ a feature of ~he primary ~netic mechanism Rind:x,A---'P"O n the olher hand, Vf may be grea!e~ ol
and {d) Snbslrate ~ynerg~sm is occurring. less ~ a n Rraax ' A ~ P depending ,~pon the ~ehtJons,hip
When considering the rationale behind slow partial between k2, k 3 and k 7. Shnfla~ argmmen~scaa be made
exchanges the fizst three explanmions are obvious and do with reference ~o the Vf and Rrna~" B~,-~Q exchange.
no~ wat~am additional explanation. Subst~ate synergism, Although no d~rect ~ela~ons~hip exi-z~sber~,een ~he
however, is a concept wNeh requires further dSscnssion partial exchange rotes aud fl~e kfifiat reaction ~elocifies,
in ~his contexL Th~s idea was first proposed by Bridget 1he following equ~lion pertains,
e~ aL ~3] ~o explain cert~n ~tow partial exchange reac-
tions ob~aLned w~th succ~yl coenzyme A synthetase. l l l I
(3)
Sh~flar slew exchanges were obse~ed wRh o~her Ping
Pong enzymes and the reader is referred to [3] for
addi~iona~ ~ f o n n a t i o n on i n s point. From ~hese considerations it is c]e~r that one mBsl
Bridget et aL [3] sugges~ thin if net reaction tales evaluale all four parameters shown in eq. 3 before
axe rapid compared with par~ia~ reactions, synergism any defin;Aiveconclusions can be d~awn regarding the
would ap0ea~ probable. These authors go on to suggesl impotence of, and explanation fol, "'slow'"p ~ a ~ ex-
that rsaction ra es (initial velocities) measured change ~cacfion% relative m i~tial ~tes.
~n the ab~.ence of pmfluet requi~ed for *he partial Another criterion ~ha~ has been used ~o evaha~e
reaction and thus a comparison between ~he two dif- subs~rate synergism ~nvoh,~s comparison of the pmfial
ferent rates difficuR ~o ~ a k e una~.~bignously. ]n- exchange reactions in the presence and absence of the
tuig~ely, one would expect in viewing scheme ] ~hat substrale-pmduct pair not intoned in ~he i s o m p k
the A ~ P an~ B ~ Q exchanges should be equal to exchange reacl~on [3]. Here i~ wouid be expected ~nm
or greater than the initial vetoci~y of ~he reaction. In- the pre~enee of B and Q would sere m decn'ase ~he
tuition in this setting appems to be misleading and no A ~ P exch~mge of scheme 1. If ~he A --~ P exchange
direct remtionship exists between hhe maximal exchange increases, ~he eoneep~ of synet~s~ may be invoked.
ra~les an~ ~he mu~Cm]atinitiM ,xeaetion velociW. These It can he. seen ~hat in the case of ~ e Ph~g Pong Bi Bi
poims are readily demonstrated as follows: mecha.~ism the overall exchangg, A ~ Q, is 3elated ~o
The late equation fo~ ~he A - - P exchange is: ~he patfia! exchanges in the p~esence of b m h substrate~
and products as follows:
1 {k2+kB) [ k l
+ -
klk3
- +
~] (2)
k2k4(P) _ _ - +__ (4)
E~ts of IJRA~_.~1' agains~ I]A at differem fixed gon-
cenI~afion~ of P W e a fam~y of paralM tines with Eq. 4 suggesls tha~ th~ partialexchange ~ate~must be
equal ~o ca"greater than the ove~sl]exchange ra~e.
and slope ~3 ]Eo. A secondary plot of ~tercepts vers~
lIP will X~e]d a ~t~g~ght line wilh intercept (k2-1-~3)]
k2ks(E0). This ~erm is equal ~o ljR{raax, A,-'P)" By 3. Oone],uion
av;alogy, one m a y rde~erm~e ] jR[max" B,..~Q), which is
e~ua] ~o (:k~,+kT)Jk6k?(E~). The p~Fos~ of flJs report is ~o pNn~ ou~.~those
T h e ~ equations reveal Lmme.diately. tha~ nO direct criteria~hich may be used to invoke sub~mte ~ n e >
le]ufions'hip ~exiStsbeiween the ,~ate of ~ e paTtiat ~x: g i ~ in o~de[ 1o a~c,o,~i~,~for "s~ow" pm%ia~ exchange -
c.,h~nge-~eac~tion~and r a a x ~ ve]oeily in either ~ e e - ~eacfiDns, t~ i~ q~file valid ~o compme p~tla] exchange
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 ~um:be:r I _ . . . . .= F]~BS-Lr..T'~'E~S.. : . " ~. .' . . ' May .~97-~

p~oduc~icompon:enl~ ,of th:e P i n g P o n g : s y s l e m a s w a g - - HomeECono~c~ Exv~rim~ni S~a~on, Am~s, l~wa :

, d o n e b y Bfi'dger .et ~al [ 9 ] , O n ~ e 0~ae,~ h e n g, it is n o t 500]0. ProjecI N0. ]665.
- p o s s i b l e 1.o !e,]a'te p ~ r ~ a t ~ex~nanges "to a~i~al r e a c t a o n " ' - . " - - . " ~ -. " " :.
veioc]~e~ in ~ n a t l c m p t t,~.expl;~dn:~o-catled.Slow ex- : ~ : : -. -
change.reac~ions~ Unfo~'tuna'te]y, t~s c : o n ~ e l ~ f i o n Js R,e:ferences - . - - .. . - . "
'to0 off'ten m a d e . " = " : " " :- " " . _ . r . ". . - . _ . .
[1] W,W. C~h~d, B i ~ h i ~ . B i ~ h y s , A c t a 6? C!963) ] 0 4 .
|2~] R.A.A.~be:tSy~ 3. A2n..,C~e~ru Soc. 7S (]953_3-~t92g.

Aeknowled~ements
70'96~) ~6'08. '
I 4 ] K. Da]z]el, A~r~a C~erm Scar~fl. 1 ~ ( 1 9 ~ 7 ) 1 7 0 ~ .
Th~s ~escarch was. supported ~n p ~ t by Resem~h [5] H A . P~romm and ]. Ning, .B~c:hem. B~ophys. Res. Com-
Gran~t N S d 0 5 4 6 ~. o m. :the
. . N a. ~ o.n a ]. ins:tat~ies" ,ofH"~ a"l ~ h , mun. 32 ~3968)672.
Uni;ted S l a t e d ~ b ] ~ c H ~ a l ~ h S ~ r v i c e , and R e s e m c h [61.] K . U3zcda,@~ B ~ L ,Ch:.m. 2 4 7 ~ 9 7 2 ) 1692.
G~-zm~t G B . 3 3 4 0 D Horn the National Science Founda- [7] .V, 2v]ass~y and B. C ~ a z d , ~: ~tn~ns.anfl fl~v,~pzo~e~n~, =d,.
K, Yag~ (U~W~zs~y Pa~k P;~s~, ~968) p. 22~.
tion. ]omma] Pape:r 3-751 :~ ,of ~ e Iowa A ~gr]cu]~me and

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