ARCHIVES OF BIOCHEMISTRY AND BIOPHYSKS Vol. 217, No. 1, August, pp. 10-14, 1982

Location of Lysyl Residues at the Allosteric Site of Fructose 1,6Bisphosphatase’

HIROYUKI SUDA,*$ GEN-JUN XU,*L ROSTYSLAW M. KUTNY,T4 PAOLO NATALINI,*v5 S. PONTREMOLI,$ AND B. L. HORECKER*”

*Roch,e Institute of Molecular Bidogy and fCh.emiud Research Department, Hoffmnn-La Rode, Inc, NutLy, New Jersey 07110, and ~Institute of Biological Ckmistry, University of Genoa, Genm, It&

Received February 18, 1982

Modification of rabbit liver fructose-1,6-bisphosphatase (EC 3.1.3.11) with pyridoxal phosphate in the presence of the substrate fructose 1,6-biphosphate has been shown to decrease its sensitivity to inhibition by AMP. Two adjacent lysyl residues, located at positions 137 and 138 in the primary sequence, have now been identified as the site of modification, each being modified to the extent of approximately 59%. These allo- steric-site lysyl residues are located in the largest cyanogen bromide peptide, designated BrCNl. BrCNl is linked to the NHz-terminal segment whose sequence was reported earlier [Botelho et al. (1977) Arch, B&hem. Bkphys. 184,535-5451. The sequence anal- ysis of this large cyanogen bromide peptide, comprising residues 80-163 of the fructose- 1,6-bisphosphatase subunit, is reported.

We have previously described (1) the modification by pyridoxal phosphate of amino acid residues at the active and al- losteric sites of rabbit liver fructose-1,6- bisphosphatase (Fru-Pzase,7 EC 3.1.3.11). This reagent reacts with residues at both

i Dedicated to Professor A. E. Braunstein on the occasion of his 80th birthday.

* Present address: Institute of Microbial Chemis- try, 14-23 Kamiosaki, 3-Chome, Shinagawa-ku, To- kyo, Japan.

* Present address: Institute of Biochemistry, Aca- demia Sinica, Shanghai, People’s Republic of China.

4 Present address: DuPont Experimental Station, Wilmington, Del. 19898.

5 Present address: Laboratorio di Biochimica Ap- plicata, Universita di Camerino, 62032 Camarino, Italy.

‘Author to whom all correspondence should be addressed: Roche Institute of Molecular Biology, 340 Kingsland St., Nutley, N. J. 07110.

7 Abbreviations used: Fru-Pa, fructose l,B-bisphos- phate; Fru-Passe, fructose 1,6-bisphosphatase; BrCN, cyanogen bromide; PTH, phenylthiohydantoin; HPLC, high-performance liquid chromatography.

sites, but can be made selective by car- rying out the reaction in the presence of either AMP, which protects the allosteric site, or Fru-Pz, which protects the active site (2). Modification of the AMP-binding site(s) results in decreased sensitivity to inhibition by AMP (1,2) as well as loss of binding of the enzyme to blue-dextran Se- pharose (1, 3). The latter property can be employed to separate the fully modified Fru-Pzase tetramers from forms contain- ing one or more unmodified subunits (1).

The residue modified at the active site has been identified as N’(phosphopyr- idoxyl)lysine (1,2) and the sequence of the cyanogen bromide peptide containing this lysyl residue has been determined (4). This cyanogen bromide peptide is derived from the COOH-terminal portion of the poly- peptide chain and the active site lysyl res- idue has been shown to be located at pos- ition 54 from the COOH terminus.

The modified residue(s) at the allosteric site were shown (1) to be contained in the largest cyanogen bromide peptide (BrCNl).

0003-9861/82/090910-05$02.00/O 10 Copyright 0 1932 by Academic Press, Inc. All rigbta of reproduction in any form reserved.

ALLOSTERIC SITE OF Fru-Pzase

C3b m-F-9

FIG. 1. Sequence analysis of BrCNl. The portions of the sequence determined by analysis by HPLC of the PTH derivatives, including the automatic sequence analysis, are indicated by the symbol -. Portions of the sequence established by identification of the PTH amino acids and also by analysis of the amino acids released by hydrolysis of the PTH derivatives, or by the subtractive methods, are indicated by the symbol -. Residues determined by hyrdrazinolysis are indicated by the symbol -. The p(phosphopyridoxyl)lysyl residues are indicated by the asterisks.

The sequence of this peptide, containing 84 amino acid residues, has now been de- termined, except for a small segment be- tween residues 38 and 49, which is iden- tified only by amino acid composition. Modification involves two adjacent lysyl residues, located at positions 58 and 59 in BrCNl, each of which is modified to the extent of about 50%. We have also isolated an overlapping tryptic peptide containing the NH2 terminus of BrCNl and the COOH terminus of BrCN2, previously shown to be derived from residues 19-7g8 of the polypeptide chain (5). This identified BrCNl as derived from residues 80-163 and locates the allosteric site lysyl resi- dues at positions 13’7 and 138 in the pri- mary sequence.

EXPERIMENTAL PROCEDURES

These were as previously reported (1, 4) or as de- scribed in the Miniprint Supplement.

s In the present work we have identified an addi- tional serine residue located at position 75. This ex- tends the sequence of BrCN2 to include residue 79, rather than ‘78 as previously reported (5).

RESULTS AND DISCUSSION

Labeling of the allosteric site with pyri- doxal phosphate and isolation of the labeled cyanogen bromide peptide. This was car- ried out in the presence of 5 mM Fru-Pz as previously described using NaB3H4 to label the reduced Schiff base (1). The la- beled protein was carboxymethylated and cleaved with cyanogen bromide and the largest cyanogen bromide peptide, con- taining most of the radioactivity, was iso- lated by chromatography on Sephadex G75 (see Fig. 3 in Ref. (1)).

Sequence analysis of BrCNl. The peptide was carried through 36 cycles of auto- mated Edman degradation (see Fig. 1 and the Miniprint Supplement). The remain- ing sequence was established by analysis of peptides isolated after digestion with clostripain or trypsin or after hydrolysis with dilute acid as detailed in the Mini- print Supplement and summarized in Fig. 1. Difficulties were encountered in the se- quence analysis of the segment containing residues 38-49; for this segment only the composition is known.

12 SUDA ET AL.

Location of BrCNl. The isolation of an overlapping tryptic peptide containing Met7’ established the linkage of BrCNl to BrCN2; the latter was previously shown to be derived from residues 19-79 of the polypeptide chain (5). The sequence of this tryptic peptide was determined to be Lys- Leu-Asp-Val-Leu-Ser-Asp-Val-Met- Leu-Lys, including the last nine residues of BrCN2 (5) and the first two of BrCNl (for details see the Miniprint Supple- ment). The inclusion of serine corrects the sequence reported earlier (5) but is con- sistent with the amino acid composition reported at that time for the correspond- ing tryptic peptide (see Peptide TZ-d in appendix Table 2 of Ref. (5)).

The conclusion that BrCNl probably fol- lows BrCN2 in the polypeptide chain was anticipated from results obtained with the pig kidney enzyme by Marcus et al. (per- sonal communication and Ref. (6)). In pig kidney Fru-Pease, methionine-79 appears to be absent, and a large peptide, corre- sponding approximately in size to the sum of BrCNl and BrCN2, is generated during cleavage with cyanogen bromide (Peak I in Fig. 1 of Ref. (6)).

Location of the allosteric-site lysyl resi- dues. The location of the allosteric-site ly- syl residues was established during the sequence analysis of the radioactive pep- tide Pal (see Fig. 1 and the Miniprint Sup- plement) isolated after hydrolysis with 0.03 M HCl. The radioactivity was re- covered in approximately equal amounts

in the ethyl acetate extracts containing the PTH-amino acids in steps 9 and 10. Free lysine was also recovered at each of these steps after hydrolysis of the PTH- amino acids, indicating that each residue was converted to the NG(phosphopyr- idoxyl)lysine derivative to the extent of about 50%. The presence of at least one unmodified lysyl residue at the allosteric site is consistent with the previous finding that sensitivity of the modified enzyme to inhibition by AMP is reduced but not com- pletely abolished (1).

ACKNOWLEDGMENT

The Institute of Biological Chemistry of the Uni- versity of Genoa acknowledges support from the Ital- ian Consiglio Nazionale delle Ricerche.

REFERENCES

1. Xv, F.-J., DA’ITA, A. G., SINGH, V. N., SUDA, H., PONTREMOLI, S., AND HORECKER, B. L. (1980) Arch. Biochem Biophys. 210,98-103.

2. COLOMBO, G., AND MARCUS, F. (1974) Bimhem- i&?-g 13, 3085-3091.

3. CRUZ, Z. M., TANISAKI, M. M., EL-DORRY, H. A., AND BACILA, M. (1979) Arch. Biochem Bbphys. 198424-433.

4. Xv, G.-J. NATALINI, P., SUDA, H., TSOLAS, O., DZUGAJ, A., SUN, S. C., PONTREYOLI, S., AND HORECKER, B. L. (1982) Arch Biochem Bie phys. 214,688-694.

5. BOTELHO, L. H., EL-DORRY, H. A., CRIVELLARO, O., CHU, D. K., PONTREMOLI, S., AND Ho- RECKER, B. L. (1977) Arch Biochem Biophys. 184, 535-545.

6. MARCUS, F., AND HOSEY, M. M. (1981) J. Bid Chem. 256, 12,208-12,212.

ALLOSTERIC SITE OF Fru-P2ase

NlllIPRIWT SOPPLMENT

EXPENlNENTK PwEWNES

kterl~lr. ““lCSS otlmwis. spedfled, tbs. were flwn soYrc.s pre- vlour~fied (51.52).

P.tiWdr . Automated rqwnc. analyrlr MS perfomed with . Beckman Mel~pinning cup s.quen.tor. ur,ng gectman pmgren .-I. 030176 IO.1 n 0"edrc.l *Ith C0lbln.d 51 and 52 werh,. ,d.nt,f,c.t,on of PTH-,rn,lm kids wds "Me with . n0dlfl.d Yeterr Hi& ierfomenc. Llqu,d Chronvtogrepby .pp.r.t"r, kdel 410. llanv.1 seq"."c. enelyris (52) end pr.p.r.tton end lsolrtion of CY.MZ" br.ml& wpt4d.s wz?. es reported eerlier (51). Hydmlyris of PTH amino add d.rl"ati".r to the fv.. amino adds was cwrled wt as described by Nendsz and La, (53)

RESULTS

Autoaeted s.a~."c. .ns,vrlr. Thirty-six cycles of autorated sequence analy~ the PTH-&no ecld derlvetiver yielded the N"2-t.ninal r.q"enc. Phom in Fig. 1. The S.W.IIC. of the first nln. reeldues '..I confirnad by ma"u.1 Edna" degredetio" of g,.ZW,. The sequence for r.Sid".l 18 to 24 wes also confirmed by menu.1 E&+." degredetion of peptlde Pa2 (se. F,g. 1 and beh).

A".ly~i~ of WPtlder produced by dleertion of MN, ,,lth clostr,P.l".. Olgertlon of ErCNl with clostripein and repmtlon of tk peptides on Sephr- dex 650 yi.ld+d 5 fluomrc.mlm-posit,". peeks (Fig. S,). peek 5 conteined NH3 and free am,"0 acids. tb.. other four yielded paptlder C,-C4, accounting for all of the nsiduer In MN1 (Table S1 end text Fig. I). Ntne steps of manual Edmen de redrtlon terminus. P

Identified peptldc Cl es dwived fmn the NH2- Pept de C3 ves tb. only peptide conteinlng hcmoserim end VII

therefore &rived fm. the COO+teminus of BrCNl. This p.ptW *es cerried thmugh three steps of menu.1 Edun degredetion. peptld. C3 wes also digested w,th tryplin (se. Nethods) end the tryptic peptlder Cla end CSb. derived fm the NH2 end CW"-tsmi"., portions of C,. respectively. were recovered end enelyred by menu.1 Edmen degredetion (se. text Ffg. I).

Anel~sis of PePtlder foti by d‘eestion of BrCNl with trywin. The p.pt1d.s forrd by dfwrtlon tith typsln (se. ".tbw,r) wem r.Parat.d by "PLC "sing a gredient of .c.tOnitril. fm 0 to 60 wrcent. Two sm.,, peptides. all s.pW.t.d fmm the others. hu,. ?.CO".r.d (deta not show,) end enelrzed. PePtlde T1 WI found to hew e coenosltlon idmticel to thet of CS. ilebl. SIi. This *es confinnd by thm steps Of E&en d.gnd.t,on. Peptld. T2. containing qrginim. was assigned to the CWH-temlnur of Cl.

13

01 I I I 100 120 140

Fraction number

Fig. 51: Oipostlon wfth clortriP.in end r.P.r.tlon of ptlder. BrCNl (10 ng)~lwo-h"ndr.d "9 of cloStrip.in (E.,.hrin~.rl. ~mvio~sl~ lncubeted In the seme buffer contalntng i0 M diihioth'mltol ena 50 nW CeCI2. wes edded and dlgcrtlon cerrled o"t et WC for 14 h. The .Ixtw. we1 soncentreted et I nl in . besl;Nn Sped-"ec ."d epplled to . SePhed.". 650 (1.6," 200 01) col~nn. pnvio~~ly egulllbrated ~4th 0.2 N W HCOj,

t pN 9.0. Elutlon was at the rete of 0.t nllmln.

The fract on "olme was 2.4 ml. Allqwts wms hydrolyzed with 0.5 N NsON for 20 "14" et lZO*C. neutralized. end e"e1yz.d for total anlno acid content with flwmscemin.. Frectlons rn pwled es indicated by the be%.

since it contained the residues left after sequence enelyrlr of psptld. P.1 (se. kla). The seq".nc. of 72 ties .rtabllrh.d by "an",, Ednun degredetlon (Fig. 1).

TABLE PI

Conposition of Peptides 0bt.in.d by Enqmt,c Digestion end Mid Acid Treetment

mptidera Pal P.2 Pa3 Cl. c3b Tl T2 T23

eRec0"er.d by HPLC es deswlkd In tha ,Uniprlnt Supplant.

b Sun of hw.,r.ri". end bws.~l". lecton..

'The "al".r ere c.1c"l.t.d besad on the retior to the q".ntlti.s of the rerlduer underlined.

14 SUDA ET AL.

t I 1 I c so 100 150

Time (mid

Fig. 52: S.p.rrtfon of cw,tid.s fomd by hydrolysis rfth 0.03 M "Cl. Ofg.stlcw and separation by "PLC ,,en c.rvf.d ."t .s d.scrfbM In ktbods. P.ptid.s labeled Pal. P.2, and Pa3 wr. an.1yz.d.

P,ptld.s form.d bv r.rtf.1 hydrolysfs with 4cfd. "ydrolysfs of'BKN1 at 105'C with 0 03 II "Cl md s.p.r.tfon by HPLC y1.fd.d . large n"llb.r of pgtfdes (Ffg. i2). includfng PI,. which contlfned the bulk of tb. r.dio- act,"fty. I(.""., Cdl" degr.d.t,on of this pgtfde ylcfded th. saqwnc. shwn In Ffg. 1. wfth th. larg.st quantfti,s of r.dfo.ctfvfty n1.as.d. in newly equal .I)".ts. (It steps 9 md 10. tiller quantftfes of r.diMctivfW ncovend In the s"cc..d,.g sups nry nfl.ct incml.te sxtractfon of the nl.tfwly po1.r PTH-16(phosphorylpyrf~4l)lysine &rfv.tiv.s. The tot.1 radfcactfvfty recowrad in th. .thyl acetate .xtl.ct MS 69%. with 281 mining in the ffn., rqwous extnct. For" wrc.nt as mcovend fn steps 9 and 10. PM-lysfn. WI also rxov.r.d In "..rly .q".l .mW"ts In steps 9 .nd 10 of the Ed"." d.gr.d.tfon, fndlcatfng that sfther of these two lysyl nsfd"w, but not b&b. ..I dwivatfzed In the relctfon ufth gyrldox.1 phosghot..

kn"., s.q"."c. analysis of ptptfd. Pa2 showed it to be d.r,v.d frcm r.sfd".s 18-33 of WC", and conffnd this pwt of the ww.nc. based on the ."tc."t.d s.q".nc. analysfs.

Peptide Pa3 "IS also subj.ct.d to ~nu.sl Ednon d.gr.d.tfon. Three steps yf.1d.d the segwnc. Pm-Lou-V.1 and estrblished the overlag bet..."

Lye Lou Aep Vat Lou Ser Asp Val Met Lou Lyr

this peptide and tlostripain psptid. C2. Howwsr, sequent. analysis f.fl.d to pvoceed beyond the thfrd stop.

Ordering of the wptides In 9rCRI. TM gnsenc. of radioactivity in peptiac ~4 sstablfshed its ov.rlap &h pptfd. P.1 and g1.c.d It on th. COO"-tenfns, sfde of C2. P.pt,d. C2 had alnrdy Le." sb0.n to follw pegtfd. Cl. b.s.d on ti,. resvlts .f ."tcatod s.q".nc. .n.lysfs. 5.q"e.c. .n.lysfs of peptfds Pal l stab1,sh.d it .I ovcrlapgfng g.ptid.s CZ md C4. th. 1.tt.r cont.,n,ng the radfaactfv. Nd(phosphopyridoxyl)lysyl residues. Bawd on th. seqwnc. analysfs of P.1 and tb. ~posftfon of C4. pcptfd. 72 WI p1.c.d at th. CWH-t.m,n"s of C4. Altbwgh no o"er1.p !,.s .".flabl. for oeotldes C4 snd C3. the ~resanc. of nrthfonfne in C3 and the .ssi.mnt of t&other peptldes icft this .s the only possfb,. .rr.ng.m."t.

Location of WNl in th. "rfmwy s.q".nce. Th. tryptfc pptfdes contafnlng rrthfonfn. ,ew located by conparing th. WLC patterns 0bt.fw.d before and .ft.T tr..t.ant of . tryptfc d,g.st of Fru-P2.s. "fth cy."og." brcmfd.. Al, of the psptfdes absrnt from the cy.~) m bronldc-tr..t.d dfgrsts ydpe ."a, 4 T23 (se. Table Sl 'i

red for th.fr .mino .cid conposft on. On., d.rign.t.d y,.,&d th. cc.wsft,on predicted f.r the overlspgfng

s.gas)lcnt contrfnfng tk CWH-tstmfnus of 8rCN2 Is.. S4) .nd the first two ""2~ten,",, r.sfd".s of ErC,fl. The s.q".ns. of this peptfd. ws .st,b,fsh.d by five st.gs of man"., Ma.. d.grrdatfo" .nd by rnrlysfs of peptfdes formed by p.rtf.1 .cfd bydmlysfs (Fig. 53). Six pptfdrs. fd.ntfff.d on the basis of tbefr lnfno rcfd cwosftfon. w.?. racowred. P.ptfd. T23P.4 was subjsctcd to s.q".nc. analysis which conffmrd the 9r.s.n~. .,f lysfn. at the coon-t.rmf""s.

Ffg. 53: %qau.nc. .n.,vs,s Of th. o".rl.twfnP twgt,c o.gt,d. (723) con- t.f"f"!I rtbfollfrm-89 n. cmposftfon Of 723 1s g,v.n I" r.m. r hptfd.s T23P.4 ;nd T23P.5 e-2~ nfno? cc.W..nts r.co".N)d fmn th. HPLC s.p.r.tions.

REFERENCES 51. A". G.-J.. Oath, A&.. Sfngh. Y.N.. Sud.. H.. Po.tr.molf. 5. .nd

Honcker. B.L. (1981) Arch. gfochn. glopbys. 2&. 98-103. 52. Xu. 0.4.. Natallnf. P.. Sudr. H.. Tsolas. 0.. 0zug.j. A., Sun. S.C..

Pontnlf. 5. and H0r.ct.r. 9.L. Arch. Blochen. bfophys.. In pv.ss. 53. Mendsz. 6. and L.,. C.Y. (1975) An.,. 9foch.m. && 47-53. 54. Botalho. L.H.. El-Dorry. H.A.. Crfvellaro. 0.. Ch". O.K.. Pontm.molf,

5. and kbrectcr. 8.L. 11977) Arch. Biochem. Bfqphys. I& 535-545.