Manganese substrate complexes of phosphofructokinase studied by pulsed magnetic resonance

AILCHIVI~S

OB

ISIOCHlcMISTHY

Manganese

ANTI

BIOPHYSICS

Substrate

Department Dallas,

k%-(i~o

Complexes

by Pulsed (3r. LARRY

164,

(1973)

of Phosphofructokinase

Magnetic

COTTA;\1

AND

Studied

Resonance ROSAKU

UYEDA

of Biochemistry, The fi,Liversit?y of Texas, Southwestern Medical School at Dallas, Texas 75255 atltl Veterarla Adrninistratio~k Hospitul, ~$500 South Lancaster Rd., Dullas, Texus 75216 Iteceived

October

20, 1972

complexes between phosphoThe formation of binary, ternar,v, and qllaternary iructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance tecahniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to rontain one or two of the metal binding sites with Kd = 20 P-II and Q 5 4, and perhaps, as many as 14 binding sites with Kd - 0.8 mM and eb = 12 ZIZ2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and AI>P which results in half maximal change of enhancement is 3@100 PM and 80 PM, respectively. No change in the water proton relaxation rate was detected upon addition of frurtose6-P or fructose-1,6-bisphosphate. A quaternary complex was det.ect,ed by proton relaxation measurements upon addiCon of fructose-6-P to a reaction mixture contailling 0, r-methylene ATP, manganese, and enzyme with 50 .UMfructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.

Phosphofructokinase (ATP: u-fructose-6phosphate I-phosphotransferase, EC, 2.7. 1.11.) catalyzes the reaction: fructose-6phosphate + ATP + ADP + fructose-l ,6bisphosphate. A divalent metal ion is required in the reaction, where it presumably functions through formation of a metalATP substrate as has been suggested by kinetic (1) and isotope exchange experiments (2). In order to obtain more information concerning the function of the divalent metal ion in the phosphofructokinase rcaction, we have employed magnetic resonance techniques to examine whether binary complexes are formed between manganese and phosphofructokinase and to detect the formation of higher order complcxcs upon subsequent addition of substrates. Jones et al. (3) have shown that only a slight enhancement of the water proton 683 Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

relaxation rate is observed when the enzymc is titrated with manganese, and upon addition of ATI’ the enhancement is increased. WC have also examined the binary complex bctwccn manganese and phosphofructokinase using bot’h electron paramagnetic resonance spectra and by water proton relaxation rate measurements. Our results demonstrate a significant enhancement of proton relaxation rates when phosphofructokinase was titrated with manganese. In addition we observed the formation of ternary complexes with t,he nucleotide substrate and quaternary complexes consisting of enzyme-manganese-nucleotide-fructose-6-phosphate by pulsed nuclear magnetic resonance techniques. Data suggesting the formation of these binary, ternary, and quaternary complexes is prescnted in this paper.

684

COTTAM MATERIALS

AND

METHODS

Phosphofructokinase was purified from rabbit muscle according to the procedure of Ling et al. (4) with modification as described previously (5). The enzyme preparation is homogeneous as judged by disc gel electrophoresis and ultracentrifugation as described previously (5). The ~r,p- and p, ymethylene ATP derivatives were purchased from Miles Laboratories. All other chemicals were reagent grade and obtained from commercial sources (6).

AND

UYEDA

rate in the presence and absence of (0) of manganese. The concentration of free manganese was determined by electron paramagnetic resonance (epr) using a Varian E4 epr spectrometer (11). The binary enhancement (Q) of manganese bound to phosphofructokinase was calculated from measurement’s of t)he observed enhancement (E*) by pulsed nmr, determination of the free manganese concentration by epr, and from the relationship shown in Ey. 1 (12) : The subscripts F, T, and B refer t)o free, total, and bound manganese, re-

spectively. theory,

The enzymic activity was determined at 28°C in a 1.0 ml reaction mixture containing 50 mM Trissulfate buffer (pH 8.1), 1.5 mM fructose-6-phosphate, 1 rnM ATP, 5 rnM MgClz ,4 mM NH&I, 0.5 mM dithiothreitol, 0.19 mM NADH, and auxiliary enzymes as described by Parmeggiani et al. (7). The protein concentration was determined from the absorbance at 290 nm in 0.1 N NaOH and the absorption coefficient of 1.09 1 g-l cm-’ (8). A molecular weight for phosphofructokinase of 380000 (1) was used in all calculations. Prior to the magnetic resonance measurements the ellzyme (0.5 ml) was dialyzed twice overnight against or Tris-sulfate (pH 8.1). 1 liter of 0.05 M Tris-HCl Tris-sulfate was used as the buffer in most experiments because the enzyme is completely stable in this buffer, while in Tris-chloride the protein is denatured readily. All of the experiments have been performed at pH 8.1 to minimize the interaction of ATP or its metal complex at the regulatory site. Initial attempts to examine similar intermediates at pH 7.4, where phosphofructokinase is allosterically inhibited by ATP, have been unsuccessful because of precipitation of the enzyme at high protein concentration.

Magnetic Resonance Measurements The longitudinal relaxation rates of water protons were determined using the 180”-90” pulsed nmr method of Carr and Purcell (9) with a Nuclear Magnetic Resonance Specialties PS 60.AW pulsed spectrometer operating at 24.3 MHz. The temperature of the samples, 50-100~1 total volume, was maintained constant (+0.5”C) during the relaxation rate measurements by using a stream of dry nitrogen and a Nuclear Magnetic Resonance Specialties P-128 Variable temperature accessory. The enhancement of the observed relaxation rate is defined a.s E* = (1/2’1~*)/(1/2’1~) (10) where l/!Z’lp is the paramagnetic contribution to the relaxat,ion rate in the presence (*) and absence of enzyme. The paramagnetic contribution to t,he longitudinal relaxation rate is I/TIP = (l/Tl) (l/T,(,,), the observed longitudinal relaxation

For a more extensive

application,

discussion of

and interpretation

of nuclear

spin relaxation measurements on biological terial, see references 13 and 14.

t* = [MnlF/[MnlT -t ([MnlB/[MnlT)@.

ma(1)

RESULTS

Intelaction

of Manganese fructokinase

with

Phospho-

Figure 1 is a Scatchard plot (15) obtained by measuring the concentration of free manganese by electron paramagnetic resonance in solutions containing 30 phf phosphofructokinasc and total manganese concentrations from 50 ~cL~Z to 1 my (Fig. 1, solid lint). These results show a biphasic curve suggestive of at least two types of binding sites. There appears to be one or two binding sites per molecule of enzyme (MW = 380 000) which have a Kd = 20 I.IRI and a second set of binding sites which correspond to -14 sites per molecule of enz.ymc with a & - 0.8 rnbl. The kinetic

activator

constant

for

mangaricse

(130 NM) was detcrmincd using a Lincweaver-Burk plot of the initial velocity mcasuremcnts with the conditions dcscribed in the Alatcrials and A,Iethods PXcept that various manganese concentrations were used instead of 5 ml,1 magnesium. When pulsed nuclear magnetic resonance was used to examine the interaction of manganese with phosphofructokinasc, we routinely observed 5-6 fold enhancements of the water proton relaxation rates. The binary enhancement (Q,) was calculated from the observed cnhancempnt trf the water proton relaxation rate (**), the concentration of free manganese measured by electron paramagnetic resonance, and the relationship in Eq. 1. The values of Ebwcrc obtained as a function of the number of

d__-

--e--

- 12 a

,h fb (A) -8

2

4

My+/

6

8

Protein.,.

FIG. 1. A Scatchard plot of the interaction of manganese with phosphofructokinase. The reaction mixtures rontained 29 pM enzyme, 50 mM T&sulfate buffer, (pH 8.1) and concentrations of manganese from 10 pM to 1 mM. The concentration of free manganese was determined by electron paramagnetic resonance. Temperature was 25V. The values of binary enhancement (A) were calculated from determination of free manganese by electron parsmagnetic resonance and the observed enhancement of the water proton relaxation rate.

manganese bound to ~h(~sphofru~toliiI~ase and are illustratctd in Fig. 1 (dotted lint). The value of q, is not constant and increases from about 4 to 10 as the r&o rtf manganese bound per pf-1ospltofructokinasc increases from 0.2-2.0, suggesting different types of manganese binding sites. Bctwcen t,he values of 2-7 m&s of manganese bound Jwr molt of protein, the binary enhancemcnt, is relatively wnstant with an average vdu(? of 8b = I:! f % from 14 scparatc dcterminations. L+2gure 2 illustra~tw the> change in obscrvcd exlharl~(~rneI~t of thtl water proton relaxation ratw in solutions of phosphofructo~ir~as~~ which wntain various concentrations of mangancwc, The results show an enhanced proton r&sation rate (t* of 3-5~3) when cneymc: is prcscnt suggesting manganese binds t,o that tnzymc. This is in contrast to thcr results of .Joncs el nl. (3) ~\:ho reported only a slight ctnhancement (Q = 1.6-2.3). This differcncc could be due to differcnccs in pH and components in the reaction mixture, sine0 thclir clxpcrimcnts were carried out, at pH 7.5, whereas ours were performed at pH S.1. To rule out t~bc ~ossi~)ilit.y t,hat the observed manganese binding to the enzyme is a result of bound nucleotidcs, the, enzyme was treated wit,h acid washed charcoal prior to the txpcrimcnt~. The results

r

1

6 0 ---

I_ 0. I

[Mn],

fmM)

lo

Fro. 2. Semilog plot of t’he enhal~eemel~t of the water proton relaxation rate by solutions containing phosphofr~~ctokinase and manganese. The solutions contained 50 mu Tris-sulfate buffer (pH 8.1);29pM (O),%PM (0),22w (A), 17rcM(A), 16 PM (II) phosphofructokinase; and the noted concent,ration of manganese. Temperature was 26°C.

obtained with the charcoal treated enzyme were identical to those shown in Fig. 2, indicating t,hat the observed enhancement is not due t,o intera~t,ioI~ of manganese with bound Ilu~lcotides on the enzyme. The possi~)ilit~ that the observed enhancement is due to binding at a site other than the catalytic site, cannot, be ruled out; hourever, phosphofructokinase inactivated by treat-

686

COTTAM

ment with iodoacetamide shows results similar to Fig. 2. Younathan, Paetkau, and Lardy (16) have previousIy shown that ATP-Mg protects phosphofructokinase against inact’ivation by iodoacetamide.

Addition of ATP to reaction mixtures containing manganese and phosphofructnkinase results in a large increase in the observed enhancement with the maximum enhancement occurring when the molar ratio of ATP per mangancsc approaches 2-5 (Fig. 3). A large excess of ATP results in a decrease in the observed cnhanccment. Since the dissociation constant for the ATP-Mn complex is 1.4 i 0.6 X lo-” M (17), and considering the n and Kd values for Mn-phosphofructokinase (Fig. l), under the conditions of the three experiments with high mangan~e concentrations, virtually all of the ATP added would be present as the ATP-Mn complex. Thus, the

E

AND

UYEDA

results shown in Fig. 3 suggest t.hat ATPMn interacts with phosphofructokinase to form a ternary enzyme-ATP-Riln complcx. It is interesting to note that the ATP concentration required for half maximal enhancement shown in Fig. 3 increases from 30 PM to 100 ~.lnr as the manganese concentration is increased. Since one might expect the half maximal ATE’ conccntration in Fig. 3 t*o remain constant if phasphofructokinase has only a single type of binding site for this substrate and the sites are noncooperative, thcsc results suggest that phosphofructokinasc has more than one type of binding site for t,hii metalnucleotidc under conditions whore more metal-nucleotidc is formed. Kemp and Krebs (18) have reported that there are at least 3 types of ATP-h’lg binding sites at pH 6.95 by gel filtration techniques. Jones et al. (3) also suggested at least two types of ATP-hln binding sites on the enzyme bascld on their magnetic rcsonancc measurcmcnt’s at pH 7.5. The &?rtlary ~?nha~~~~rn(~nt value obtained for the ATP-Mn-phosphofructokinasc complex was determined as a plot of 1/t,* vs l/[protcin] (19) from oxpcriments which conta,ined 0.33 m&f ATP, ma.ngancse, and variable ~o~~centrati~)~~s of phosphofructokinasc. From t,hree separate experiments similar to t’hc one shown in I?ig. 4 the value of 6, of approximately 12

r

IQ

100

[ATP] (NM) FIG 3. Sernifog plot af the effect of ATP on the observed enhancement of phosphnfructokinasc and manganese. The conditions were : 50 mM Trissulfate buffer (pH 8.1), (A) 34.8 NM enzyme, 170 pM manganese; (e) 34.5 pM enzyme, 360&M manganese; (H) 11.6 prd en.3yme, 330&x manganese; (0) 30.8 pM enzyme; 14 pM manganese; (A) 10.5 pM enzyme, 14 BM manganese; and the noted concentration of ATP. Temperat’are was 25’C.

01

0.1 I/Protein

0.2

(ml/mg)

FIG. 4. Titration of ATP-Mn with phosphofrue~ak~nase. The resetion mixtures contained 50 mnz Tris-s&fate buffer (pH 8.1), 0.30 mM manganese, 0.51 mM ATP, and the noted concentrations of protein.

l’~IOSPHOFl~UCTOKINAsE

Mn-SUBSTRilTE

was obtained. Numerous attempts to fit this data by computer programs consideriug all possible complexes that form have thus far been unsatisfactory. When ADP was used to titrate solutions containing 22 PAI phosphofructokinase, lG7 ~41 manganese and 50 ml1 Tris-sulfnk buffer, pH S.1, the observed enhancement increased as t.hc ADP con~eI~tration increased. The maximal cnhaSneement value observed was twice the value in the sbwnce of ADP, and the concentration of ADI’ at half maximal change in enhancement n-as so p\i ,4DI’. The possible forrnati~~I1 of ternary tom-, plcxes with either &k-G-P or Fru-1, G-bisP and the Mn-phosphofructokinasc complex were also invc&igated and t*hese results are show11 in Table I. The addition of either Fru-fi-P or J’ru-1 ,G-bisP to a solution cow tsining ~n~~Ilgar~es(~ and ~~hosphofrLl~tol~irlas~ results in only a slight (
COMPLICCES

687

phosphofructokiIlsse complexes wre examined, and the results art: shown in Fig. 5. dccrcasrs In both cases swn in Fig. 5, l/T,,* as the absolute tttmperaturc increases. Since l/2;,* leas a negative tcmpcrat,ure w&icient, rw is not bhe rat,e limiting prorcss as discuswd by JIildvan and Cohn (13). i~urthcrmorc, the c~nhancemcnts of tht water proton relaxation rates are much $IYv%tcY tllarl 1.0 (e* = J-12; Et = 12); thus the relaxation rate measurement,s are not primarily a result, of XI out,cr coordination sphere relaxation process (13). In order to rule out the possibility that, the observed effect on l/TIP* is a result of manganese dissociation from the various enzyme complexes, the conccntrat,ion of free mangancsr at various temperatures from l:iWiT,“C in increments of 10°C was measured using electron paramagnetic rcsotrance. In reaction mixtures ~o~~t~aining 20 ~BI enzyme, 77 PM manganese, and 50 rn>r Tris-sulfate buffer (pH X.1), and 77 par ATI’ or 77 P\I @,y-mcthylerw ATI’ and

and Ternaq

The effect of tJcmpcrature on the water proton relaxation ratt measurements Gth girt-pIlt,sI.lhofrrrctokinaae and AIn-ATP-

0 7.7 20 29 43 100

3.Y 3 Y 3.7 3.7 f‘3”., 3.5

0 28 50 so 200

3.8 3 0 3.6 3.5 3.5

*The solutions contain 50rnLt Tris-sulfate (pH R.l), 83~~ manganese, 2-1PM phosphofructokinase, and the given concentrations of fructose-fiphosphate or fruclose-l-6.bis:~hosphate. Temperature was 25°C.

FIG. 5. Effects of temperature on the paramagnetic contribut,ion to the water proton relaxation rate in solutions of Mn-phosphofructokinase Mn-ATP-phosphofructokinase (e--O) and (O---O). The solutions contained 50 mns Trissulfate buffer (pII 8.1), 24~~ phos~hofr~~ctokinase, 83 p&f manganese, and (0 - 0) 83 N*MATP.

688

COTTAM

AND

UYEDA

77 PM Fru-6-P, the concentration of free manganese remained constant over the entire temperature range at 44 plcp, 18 FM and 9 ,UM, respectively. The interpretations of the relaxation rate measurements described here are therefore based on the conclusion that the relaxation mechanism is a dipole-dipole interaction in the first coordination sphere. Formation oj Quaterna y Complexes Proton relaxat,ion rate measurements were performed in an attempt to detect the formation of a quat,ernary complex consisting of ADP, enzyme, manganese, and &u-l, &bisP, t,he reversal of the phosphofructokinase react,ion. As the ADP concentration is increased from O-77 PM in a solution containing 22 jails enzyme, and 77 PM l’ru-1,6-b% t’he observed enhancement increased from 3.0-4.4. This increase in observed enhancement is slightly less than that observed in the absence of Fru-1 ,6-bisP. These results would be consistent with formation of a quaternary complex. In order to obtain further evidence fol the fornlation of a q~laternary complex, the methylene analogs of ATP were tested. Kinetic studies have shown t,hat the ~11 ,/Lmethylene analog of ATP serves as a substrate, whereas the /3, y-methylenc analog is a competitive inhibitor of ATP with an apparent inhibitor constant (Iii> of 0.7 mar. When examined by wat,er proton relaxation rate measurements, addition of the p, y-methylene ATP analog to phosphofructokinasc and manganese results in an increase in enhancement suggesting formation of ternary complex, but higher concentrations of the analog are required than with ATP. Since p ,r-methylene ATP forms a ternary complex with manganese and enzyme, but cannot transfer its terminal phosphoryl group to Fru-6-P, we attempted to demonstrate the formation of an inactive, quaternary complex consisting of the 8, y-methylene ATP, Fru-6-P, manganese, and phosphofructokinase. The results, as shown in Fig. 6, suggest the formation of such a complex. The observed enhancement decreases from 3.9 to 2.9 upon iilcreasing the Fru-6-P concentration up to 1 maI in a

FIG. 6. Semilog plot of the observed enhancement of the water proton relaxation rate at various concentrations of Fru-6-P. The solutions contained 33 rnM Tris-sulfate (pH S.l), 83 pod manganese, ~~MIVIphosphofructokinase. 92~~ fl,r-methylene analog of ATP and the noted concentrations of Fru-6-P. Temperature was 22°C. The point on the ordinate contains no Fru-6-P. The inset illustrates the effect of temperature on the observed l/Tip* values for R solution containing 33 mM Uris-sulfate (pH 8), 17 FM phosphofr~~etokinase, 93 p&t P,r-methylene ATP, 83 PI% manganese, and 83 PM Fru-6-P.

solution containing manganese, phosphofructokinase, and the @,y-methylene ATP. No decrease in enhancement is observed if the ~,~-meth~lene ATP is deleted from the reaction mixture. The Fru-6-P concentration that gives half maximal change in the observed enhancement in t’he quaternary complex titration (Fig. S), is 0.04 mar which is in close agreement xv&h the ~~ichaelis Constant of 0.05 ml1 for Fru-6-P obtained from kinetic experiments using ATP under similar experimental conditions. The effect of temperature on the paramagnetic contribution to the relaxation rate of the quaternary complex is seen in the inset of Fig. 6. The relaxation mechanism apparently is a result of a first coordination sphere relaxation process as discussed for Fig. 5. The decrease in enhancement observed with the increase in Fru-6-P concentration could result from displacement of the MnATP analog from the enzyme. If such a

displacement occurs one would expect an observable increase in the amplitude of the cpr spectra. This possibility seems t,o be ruled outs because no increase in an~plitude of t)hc cpr spectra was d&&d upon addition of 16 mu E’ru-6-P to a solution contaiuing 3:j m.u Tris-su1fat.e (pH %I), 83 q manganes(~~ 92 ~.tnr 8 ,~-n~etll~le~~e ATI’, and 14 ~.r~i pllosphofructoliinase. ‘However, the observed dccrcasc in the enhancement values upon addition of Fru-6-Z’ (Fig. 6) wuld hc c~xplaintd by slight changes in the rclasation mechanism. DISCUBSION

The data presented here demonstmtc an interaction of manganese with phosphofructokinase. The analysis of the Scatchard plot, (Fig. 1) reveals two different binding sites on the enzyme for manganese. One of the sites is probably at t,hc catQ+c site evrn t’hough the dissociation constant (20 ,L,I) is somclr-hat lower than the kinetic activa,tor constsnt (130 PM). The role of thci other manganese binding site, with a dissociation constant, of 0.S mlr, is not clear at, present, hut could br a nonspecific site. In contrast8 t$o our results, Jones ef al. (3) reported t,hat low cnhanccmttnts (Q = 1.62.3) wcw obtained from manganese titrat,ions of phosphofructolrinxsc. The rcason for the discrepancy bct~vecn their values of 6’0and our values of If! f 2 arc not obvie IUS but could be due to diffwenws in c~xperimental proccdurw used. WC dialyzed the cnzymc ~~xhaust,iv~~l~ to remove any residual amount of EDT:2 which was present in the stock solution of the enzyme, ~hcreas their dialysis \YMSonly for 2 hr and might have ~ont:~i~~~~~~ a f~rattc amount of EDT,%. In addition, all of our nmr st,udies were done at pH S.1 in Tris-sulfate buffer while they used Tris-HCl at pH T-0 and 7.5. Sones et nl. (3) have reportc~d binding of Bin-ATP to phosphofructotiirtasr? at pH 7.3 and, since et > fb, suggcstcd t’hat the ternary complex is a substrate bridge complex. In our present studies w also obscrvcd a large increase in cnkancement n-hen the ATP concentration was increased in reaction mixtures containing a wnstant concentration of protein and mnngarwse. Howver,

we observe Eb = 12 f 2 which is larger than the little or no binary enhanccmcnt gonerally found Tvitb substrate bridge enzymes, or Type I complexes (13). If be wnsider only the tighter manganese binding site (Q < 4), then we would have the situation Ed > Emwhich is consistent with a sub&r&e bridge con-&x. However considering the weaker manganese binding site, we observe E, = Edwhich is similar to results seen with Type III, the enzyme bridge complex of subst.rate-e~~zyrne-mienganese (13). Our results could also be consistent

with

a. 15<‘I’

. Tape

IT

complex 4 ,

since a metal binding site is present both in the presence and absence of nuclcotide substrates. Since we do not know the role(s) of two separate binding sites for manganese, we cannot conclusively decide on the t,ype of coordination scheme for the manganesenucleotide-phosphofructokinase complexes. Ternary complexes were swn with both nucleotides ATP and ADY, but, could not bc detected between l?ru-B-l’, manga.nese, and phosphofructokinase. These results are similar to Type I enzymes when the nuclco~ide-rn~~~~~a,nese imcracts with the enzyme to form t,he substrate bridge complexes. ,\Zanganese-nucleotide-enzyme ternary complexes appear t,o be similar to the cat,algt,itally a&ivc forms, since the co~l~~~ltra~~jo~~s at one-half maximum enhancements of the proton rtklaxation rate mCasuremcnt,s (:iTP = 30-100 ~11, ADP = SO ,~lr> arc close to a~~~~re~~~ K, (7;i ,~ar) and Ki (130 PM> values for ATI’ and ADP, respectively, as dctcrmincd kinctieally under similar conditions (2). C+VI‘d encc previously reported (3) ~~~~rn~)nstrat,i~~~ a divalcnt metal rcyuirement for both the ATP:ADP and tho Fru-G-P: Fru-1 ,6-bisP exchange reactions is also consistent. with the postulate for two separate binding sites for divalent metal ion. At present at lcast two other enzymes, pyruvate carboxylasc (20) and phospho’nolpyrnv~~t~ synthet,asct (21) apparently require tfivalcnt metal ions for tn-o separate functions in the catalytic: reactions. X:0 ternary complex couId bc detected when Yru-1 , G-b&P or Fru-6-P were sddcd

690

COTTAM

AND UYEDA

t,o reaction mixtures containing phosphofructokinase and manganese. However, if the reaction mixtures also contained a nucleotide, ADP, or ATP respectively, the observed enhancement decreased upon addition of the sugar phosphates. The p, y-mcthylene ATP analog was then used to form the ternary, enzyme-analog ATPMn complex which could not t,ransfer its phosphoryl group to the Pru-6-P when it was added t,o the reaction mixture (Fig. 6). The decrease in the enhancen~ellt seen in Fig. 6 suggests formation of the quaternary Fru-6-P-methylene ATP-Mn-phosphofructokinase complex. The binding of the nucleotidc and manganese to phosphofructokinase are apparently required prior to or help form the site for the binding of Fru-6-P. However, decreases in observed enhancement could be due t’o displacement of manganese or methylene ATP-Mn from the complex upon Fru-6-P binding. Yet no increase in the manganese epr signal amplit,udc was detected upon Fru-6-P addition to the enzyme-bin-methylene ATP complex. Previous studies which were based on kinetic and isotope exchange experiments suggested that the mechanism of the phosphofructokinase reaction might be “pingpong” (2). This mechanism would suggest that ADP is released from the enzyme before Fru-6-P binds and implicates formation of a phosphoryl-enzyme intermediate. However, more recent. kinetic studies (22) using Fru-1-P as a substra.te rather than Fru-6-P provided evidence in support of an ordered mechanism in which formation of a quaternary complex occurs. In an attempt to obtain additional information to distinguish between these two mechanisms, sequential or “ping-pang” (23), proton relaxation rate experiments mere carried out to determine whether a quaternary complex is formed. Evidence for the formation of a quaternary complex is provided by the following experiments: (a) A decrease in enhancement was was observed when Fru-1 ,6-bisP added to a ternary complex consisting of awn-ADP-enzyme. (b) A decrease in enhancement was also observed when Fru-6-P was added to the Mn+, y-methylene ATPenzyme ternary complex. Thus, our present studies with proton relaxation rate measure-

merits provide direct evidence for a quaternary complex formation and seems to rule out a “ping-pang” mechanism. ACKNOWLEDGMENTS This work was supported by Grant I-381 from The Robert A. Welch Foundation, Houston, Texas, a grant from the American Heart Association, Texas Affiliate, Inc., and Research Grant BM 16194 from t,he National Institute of He&h, U. S. Public Health Service. REFERENCES 1. PIETKAC, V., ASD L.IRDY, II. A., (lQ67) J. Biol. Chem. 242, 2035. 2. UYEDA, K., (1970) J. BioZ. Chem. 246, 2268. 3. JONES, R., I)WEEC, B. A., AND WALKER, I. 0. (1972) Eur. J. Biochem. 28,74. 4. LING, K. H., M.~RCUS, F., AND LARDY, II. A. (1965) J. Biol. Chem. 240, 1893. 5. UYEDA, K. (1969) Biochemistry 8, 2366. 6. TJYBDA, K., AND RACKER, E. (1965) J. Viol. Chem. 240, 4682. 7. PARNIEGGIANI, 8., LUFT, J. H., LOVE, I>. S., AND KREBS, E.G. (1966) J. Biol. Chem. 241, 4825. 8. PAE~KAU, V., YOUNATHAN, E. S., AND LARDY, H. A. (1968) J. Mol. Biol. 33,721. 9. C.&RR, H. Y., AND PURCELL, E. M. (1954) Phys. Rev. 94, 630. 10. EISINGER, J., SHULMAN, R. G., AND SZYMANSKI, B. M. (1962) J. Chem. Phys. 36,172l. 11. COIIN, M., AND TOWSEND, J. (1954) il'ature (London) 173, 1090. 12. MILDVAN, A. S., AND COHN, M. (1963) Biochemistry 2, 910. 13. MILDVAN, A. S., AND COHN, M. (1970) khan. Enzymol. 33, 1. 14. MILDVAN, A. S. (1970) In The Enzymes, (Boyer, P. pt., ed.) Vol. 2, 3rd. Edit,., p. 445, Academic Press, New York. 15. /SG~T~E~ARD, G. (1949) Ann. iVY Acad. Sci. 61, 660. 16. YOUNATHAN, E. S., PAETKAU, V., AND LARDY, H. A. (1968) J. Biol. Chem. 243, 1603. 17. MILDVAN, A. S., AND COHN, M. (1966) J. b’iol. Chem. 241, 1178. 18. KEMP, R. G., AND KREBS, E. G. (1967) Diochemistry 6, 423. 19. HIMES, R. H., AND COHN, M. (1967) J. Biol. Chem. 242, 3628. 20. MILDVAN, A. S., SCRUTTON, M. C., ANT) UTTER, M. F. (1966) J. B&Z. Chenz..241,348S. 21. BEEMAN, K. M., AND COHN, M. (1970) J. Riob. Chem. 246, 5309. 22. UYEDA, K. (1972) J. Biol. Chem. 241, 1692. 23. CLELAND, W. W. (1970) In The Enzymes, (Bayer, P. D., ed.) Vol. 2, 3rd. Edit,. p. 1, Academic Press, New York.