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624
BIOCHEMICAL SOCIETY TRANSACTIONS
E-Mg-ATP-ADP(4)
E-M%-ADP
E-ADP(1)
/I
\\
E -E-Pyr-P-E-Mg-ADP-Pyr-P
I
E-Mg-
(2)
1
E-Mg-Pyr- P
(3)
I
E-Mg-ATP
/\
\ JE
E-Mg -ATP-Pyr
E-Pyr
I
E-Pyr-Mg-ADP(5)
Scheme 1. Reaction scheme showing five dead-end complexes (1-5) for the pyruvate
kinase from the leg muscle of C.maenas
Formation of quaternary dead-end complexes is shown only once for convenience.
Pyr-P and Pyr represent phosphoenolpyruvate and pyruvate respectively.
products to enzyme complexes other than those to which they normally combine in the
reverse reaction can change the form of the observed inhibition patterns. Both products,
pyruvate and ATP, were non-competitive inhibitors with respect to the substrate ADP.
This is the expected pattern if the two dead-end complexes ADP-enzyme-pyruvate and
ADP-enzyme-ATP occur in the reaction mechanism. Scheme 1 summarizes a reaction
mechanism that is consistent with both the intial rate and product-inhibition data.
It is a rapid-equilibrium random Bi Bi mechanism in which five dead-end complexes are
present.
Ainsworth, S. & MacFarlane, N. (1973) Biochem.J. 131,223-236
Giles, I. G., Poat, P. C. & Munday, K. A. (1975) Biochem. SOC.Trans. 3 , 7 1 4 7 1 6
MacFarlane, N. & Ainsworth, S. (1972) Biochem.J. 129,1035-1047
Mildvan, A. S . & Cohn, M. (1966)J. Biol. Chem. 241,1178-1193
Newton, C. J., Poat, P. C. & Munday, K. A. (1976) Biochem. SOC.Trans. 4,479-481
Reynard, A. M., Has, L. F., Jacobson, D. D. & Boyer, P. D. (1961) J. Biol. Chem. 236,22772283
Equilibrium Isotopic-Exchange Studies of the Reaction Catalysed by
Rabbit Muscle Pyruvate Kinase
IAN G. GILES, PETER C. POAT and KENNETH A. MUNDAY
Department of Physiology and Biochemistry, University of Southampton, Southampton
SO9 SNH, U.K.
Rabbit muscle pyruvate kinase (EC 2.7.1.40) catalyses a reaction between ADP and
phosphoenolpyruvate, resulting in the formation of ATP and pyruvate, which is dependent on the presence of a bivalent cation, usually Mg2+,and a univalent cation, usually
K+ (Kachmar & Boyer, 1953). The bivalent cation can bind directly to the enzyme
(Mildvan & Cohn, 1965), and it has k e n suggested that it is a true substrate of the reaction in the forward direction (Ainsworth & MacFarlane, 1973). Other workers, however, have suggested that Mg-ADP is the true substrate of the enzyme (Melchior 1965;
Cleland, 1967). Notwithstanding the ambiguity in identification of the true substrates,
all the product-inhibition experiments of the forward reaction reported are consistent
with a rapid-equilibrium random mechanism (Reynard et a f . , 1961 ; Mildvan & Cohn,
1966; Ainsworth & MacFarlane, 1973).
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563rd MEETING, LONDON
625
An assay suitable for kinetic studies of the reverse reaction of pyruvate kinase, and
based on the transfer of label from [Q2P]ATP to phosphoenolpyruvate, has been reported and used to conduct product-inhibition experiments (Giles et al., 1975). All the
inhibitionswerecompetitiveandareconsistentwitharandommechanism. Replots ofthe
slopes of the primary double-reciprocal plots (of reaction rate against ATP concentration) as a function of inhibitor concentration were linear. When pyruvate was the variable
substrate, however, non-linear-slope replots were obtained.
Non-linear-slope effects normally reflect multiple combination of an inhibitor with the
enzyme. It is notable therefore that the non-linear-slope replots were seen with both
products, but only when pyruvate was the variable substrate. This would appear to
rule out formation of a complex involving more than one molecule of the inhibitor in a
rapid-equilibrium random mechanism as an explanation of the non-linear-slope effects.
As an alternative, it was suggested that the binding (or realease) of pyruvate might not
be completely rapidequilibrium and that the reaction possesses some non-rapidequilibriumcharacter (Giles etal., 1975). This possibility has been investigated bymeasuring the initial rates of isotopic exchange at chemical equilibrium. In a truly rapidequilibrium random mechanism the rates of all the isotopic exchanges will be the same
in a given set of equilibrium conditions.
The experiments were performed at pH7.4 and 25°C in the presence of 25m~-Tris/
HCI, 1mM-EGTA [ethanedioxybis(ethylamine)tetra-acetate], 100m~-K~,,.,and 0.5
rnM-Mg:;..
In these conditions the apparent equilibrium constant of the reaction was
measured as 23500 when expressed in terms of total substrate concentrations. In all the
experiments, except the pyruvate-into-phosphoenolpyruvateexchange, the equilibrium
mixture of reactants was incubated at 25°C for 5min, after which sufficient enzyme to
give a linear exchange rate was added. A further lOmin incubation was carried out in the
presence of theenzyme to ensure chemical equilibrium. Control experiments showed that
no loss of enzyme activity occurred in this incubation. The exchange reaction was initiated by addition of a small volume (less than 1 % of the volume of the incubation mixture) of a labelled substrate. In three cases ([2-3H]ATP, [2-3H]ADP and [p3'P]ATP)
addition of the isotope resulted in less than a 0.01 % increase in the concentration of that
Time (min)
Fig. 1. Linear appearance of IabeI from [I-'4C]pyruvate into phosphoenolpyruvate as a
frrnction of time
The exchange was measured at pH7.4 and 25°C in the presence of 25m~-Tris/HCI,
1 mM-EGTA, 100rnM-K:,,,,, 0.5mM-Mg?Ae, 1m ~ - A T P ,12.9m~-pyruvate,2 5 f l ~ - A D P
and 22.1 ,u~-phosphoenolpyruvate.The pyruvate contained 0.16pCi of [I -14C]pyruvate/,umol. The exchange reaction was initiated by addition of 0.6pg of rabbit muscle
pyruvate kinase per ml of reaction medium.
VOl. 4
626
Exchange
measured
BIOCHEMICAL SOCIETY TRANSACTIONS
(I)
Fig. 2. Histogram showing the relative exchange rates for the rabbit niuscle pyruoate
kinase reaction
The exchanges measured were: (1) ATP into phosphoenolpyruvate; ( 2 ) ATP into ADP;
(3) ADP into ATP; (4) pyruvate into phosphoenolpyruvate; (5) phosphoenolpyruvate
into pyruvate. The equilibrium conditions of the unhatched parts are as in Fig. 1. Those
for the hatched parts are the same as in Fig. 1 except that the concentrations of ATP,
ADP, pyruvate and phosphoenolpyruvate were 7 . 6 5 m ~ ,5 0 p ~ 7, . 6 5 m ~and 5 0 p ~
respectively. The results are expressed as a ratio to the slowest exchange (phosphoenolpyruvate into pyruvate). The rate of this slowest exchange in the second (hatched)
condition was 66 % of that in the first equilibrium set of conditions.
substrate. In the fourth case (phosph~enol[l-~~C]pyruvate)
the increase was 5 %.
Samples of the reaction mixture were taken at various times over 6min and added to
trichloroacetic acid and placed on ice to stop further exchange. The pyruvate-intophosphoenolpyruvate exchange was initiated by addition of enzyme to a mixture at
25°C already containing isotope.
The acid-quenched samples were neutralized with Tris base and the labelled product
resolved by chromatography on Dowex 1 (C1- form). The product was counted for radioactivity by using conventional liquid-scintillation counting techniques. The exchange
rate was determined from the linear slope of the d.p.m. appearing in the product as a
function of time, and from the specific radioactivity of the labelled substrate.
Five of the six possible exchanges were measured and all gave a linear exchange over
the time-course studied, even though the zero time value may be high relative to the slope
of the line. This is illustrated in Fig. 1 for the unfavourable pyruvate-into-phosphoenolpyruvate exchange. Measuring the rate of exchange between a substrate/product pair in
either direction gave values that were within experimental error of each other, indicating
that the system was at chemical equilibrium. Further, it was found that extending the
preincubation period with the enzyme for up to 1h caused no change in the exchange rate,
again indicating the equilibrium condition. Control experiments showed that exchange
was dependent on the presence of enzyme and all reactants. This is evidence that a direct
phosphate transfer occurs.
The initial rates of isotopic exchange were determined in two sets of equilibrium
conditions (Fig. 2). The substrate/product ratios used were limited, unfortunately, by the
unfavourable equilibrium constant of the reaction and by the difficulty in removing ADP
inpurity from ATP. The data obtained, however, indicate that the phosphoenolpyruvateinto-pyruvate exchange was slower than the other exchanges, including the ATP-intophosphoenolpyruvate exchange, which has phosphoenolpyruvate as a common partner.
1976
563rd MEETING, LONDON
627
This suggests that the steps involving the binding (or release) of pyruvate may contribute
towards the overall rate of the reaction. If this is true, it follows that, strictly, it is invalid
to use the rapidequilibrium assumption to describe the rabbit muscle pyruvate kinase
reaction, although in the forward reaction all the steady-state kinetic data available are
consistent with this assumption.
Of the other kinases studied, creatine kinase is truly rapid-equilibrium (Morrison &
Cleland, 1966), whereas the sugar-sugar phosphate exchange of galactokinase
(Gulbinsky & Cleland, 1968) and hexokinase (Fromm et af., 1964) is slower than the
other exchanges by a factor of 1.5 to 2.5.
Ainsworth, S. & MacFarlane, N. (1973) Biochem. J. 131,223-236
Cleland, W. W. (1967) Annu. Rev. Biochem. 36,77-112
Frornrn, H . J., Silverstein, E. & Boyer, P. D. (1964)J. Biol. Chem. 239,3645-3652
Giles, I. G., Poat, P. C. & Munday, K. A. (1975) Biochem. SOC.Trans. 3,312-314
Gulbinsky, J. S. & Cleland, W. W. (1968) Biochemistry 7 , 566-575
Kachmar, J. F. & Boyer, P. D. (1953) J. Biol. Chem. 200,669-682
Melchior, J. (1965) Biochemistry 4, 1518-1525
Mildvan, A. S . & Cohn, M. (1965)J. Biol. Chem. 240,238-246
Mildvan, A. S. & Cohn, N. (1966) J. Biol. Chem. 241, 1178-1193
Morrison, J. F. & Cleland, W. W. (1966)J. Biol. Chem. 241,673-683
Reynard,A. M.,Hass, L. F., Jacobsen,D. D. &Boyer P. D. (1961)J.Biol.Chem. 236,2277-2283
Collagenolytic Cathepsin Activity in Rabbit Peritoneal Polymorphonuclear
Leucocytes
WALTER T. GIBSON,* DAVID W. MILSOM,* FRANK S. STEVEN* and JOHNS.
LOWEt
*Department of Medical Biochemistry, University of Manchester, Stopford Building,
Oxford Road, Manchester M13 9PT, U.K., and t Department of Biochemistry, I.C.I.
Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K.
The discovery of collagenolytic cathepsins in rat and human liver (Milsom ef al., 1972),
rat leucocyte granules (Anderson, 1971), post-partum rat uterus (Etherington, 1973)
and bovine spleen (Etherington, 1976), as well as the demonstration of the collagenolytic
activity of purified cathepsin B1 (Burleigh eta!., 1974; Etherington, 1974), suggest that
acid proteinases play an important role in collagen degradation.
Although collagenase has been found in rabbit polymorphonuclear leucocytes
(Robertson er al., 1972), there has been no report of an acid proteinase with collagenolytic activity in these cells, although Cochrane & Aikin (1966) showed that extracts of
rabbit polymorphonuclear leucocytes will release peptides from purified glomerular
basement membrane when incubated at an acid pH. This activity was attributed to
cathepsins D and E, which, in addition to cathepsin A, were detected in rabbit polymorphonuclear-leucocyte granules by Wasi e f al. (1966). No cathepsin B or C activitywas
observed. In this study, we report that rabbit polymorphonuclear leucocytes contain an
enzyme capable of degrading polymeric collagen at an acid pH.
Rabbit peritoneal exudatepolymorphonuclear leucocytes were obtained by themethod
of Cohn & Hirsch (1960). Lysates of these cells were capable of solubilizing polymeric
collagen, isolated by the method of Steven (1967), at pH4.0 and 37°C. The collagen preparations showed a small but variable susceptibility to degradation by trypsin, sometimes
exceeding 10%. Activity in cell lysates and isolated granules usually exceeded that of
trypsin on the same preparation of collagen, and, as further indication of its ability to
degrade native collagen, the enzyme was shown to be able to solubilize polymeric
collagen that had been pretreated with trypsin.
Optimal activity of the collagenolytic cathepsin was found at around pH3, but assays
were generally performed at pH4.0 to minimize the risk of denaturation during incubation at 37°C. There was no increase in the trypsin susceptibility of polymeric collagen
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