Proc. Nati. Acad. Sci. USAVol. 90, pp. 1237-1241, February 1993Biochemistry

Phosphoglucose isomerase: A ketol isomerase with aldolC2-epimerase activity

(glucose 6-phosphate/fructose 6-phosphate/mannose 6-phosphate/NMR/anomerase)

STEVEN H. SEEHOLZER*Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111

Communicated by Irwin A. Rose, August 17, 1992 (receivedfor review July 23, 1992)

ABSTRACT With 13C NMR, phosphoglucose isomerase(PGI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) isshown to produce mannose 6-phosphate (M6P) slowly from amuch more rapidly catalyzed equilibrium between glucose6-phosphate (G6P) and fructose 6-phosphate (F6P). The iden-tity ofM6P and its formation from G6P plus F6P are confirmedby IH NMR and by the ability of PGI to convert M6P to F6Pplus G6P. The possibility of contaminating phosphomannoseisomerase (PMI, D-mannose-6-phosphate ketol-isomerase, EC5.3.1.8) is ruled out by rmding no exchange of the C1 protonof G6P or of M6P, whereas exchange occurs with a mixture ofPMI and PGI in 2H20. The pro-R and pro-S protons of F6Pbecome the anomeric protons of M6P and G6P through theactions of PMI and PGI, respectively. Both isomerases ex-change the C2 proton of their substrate with the medium;hence, when PGI and PMI are added together to hexosephosphate solutions in 2H20, both the substrate and anomericprotons are exchanged rapidly with deuterons from the me-dium. The rates of C2-epimerization of G6P and M6P by PGIare shown to be proportional to enzyme concentration andinhibited by 5-phosphoarabinoate, a competitive inhibitor ofthe previously demonstrated isomerase and anomerase activi-ties of PGI. These data show that the epimerization is enzy-matically catalyzed and suggest the involvement of the sameactive site for all three activities. A primary kinetic isotopeeffect of 7.5 (H/21H) on the rate constant kC,t of the M6PC2-epimerase activity was determined by using a coupledenzymatic assay. A model of the mechanism of PGI is offered,which relates C2-epimerase activity to the isomerase andanomerase activities by allowing the cis-enediol intermediate torotate about the C2-C3 bond axis followed by protonation atC2 but not at C1 from the si face.

Phosphoglucose isomerase (PGI; D-glucose-6-phosphate ke-tol-isomerase, EC 5.3.1.9) was first found by Lohman (1) tocatalyze the reversible interconversion of glucose 6-phos-phate (G6P) and fructose 6-phosphate (F6P). Subsequentmechanistic studies with isotopically labeled medium (2, 3)and/or substrates (4, 5) revealed intramolecular proton trans-fer between C1 and C2 with partial exchange of the proton ofthe substrate with protons derived from the medium, rulingout a hydride-transfer mechanism. A single active-site basewas revealed by demonstrating intramolecular proton trans-fer, and the conjugate acid is known to be monoprotonic, asshown by transfer/exchange ratios exceeding one undersome conditions (3, 4). The steps involving proton transferwere shown to be rate-limiting when the H/3H isotope effectof F6P -- G6P was found for the isomerization reaction (3).The absolute stereochemistry of PGI has also been estab-lished (2). Thus, we know that PGI catalyzes the abstractionand intramolecular transfer of the C2 proton of G6P to yield

the pro-R hydrogen at C1 of F6P by using a single active-sitecatalytic base and a cis-enediol intermediate. Evidence forthe cis-enediol intermediate is reviewed by several authors(4, 6, 20, 21).PGI was found to catalyze a second kind of reaction when

the interconversions of the a- and ,B-pyranose anomers ofG6P (7) and the a- and l3-furanose anomers ofF6P (8) by PGIwere first demonstrated. These findings have more recentlybeen confirmed by using 31P NMR (9) and 13C NMR (10)spectroscopic techniques. The anomerase activity requiresring cleavage, subsequent torsional rotation about theC1-C2 bond for G6P and C2-C3 bond for F6P, and finallycyclization to the opposite face ofthe carbonyl plane. Studieswith epoxide-inactivated PGI (8) suggest that both anomersof each substrate react at the same active site responsible forcatalyzing the isomerase reaction. PGI was also shown tomutarotate the anomers of mannose 6-phosphate (M6P) at arate similar to its effect on G6P (11).Reported here are experiments that demonstrate and char-

acterize yet a third enzymatic reaction catalyzed by PGI:G6P C2-epimerization. This reaction results in the productionof MGP from G6P and F6P. A mechanism is proposed thatshows the relationship between the isomerase, anomerase,and epimerase activities of PGI.

MATERIALS AND METHODSRabbit muscle PGI was obtained from Sigma as a suspensionin ammonium sulfate. The suspension was centrifuged anddecanted. The pellet was dissolved in a small volume ofHepes buffer and then dialyzed against several changes ofbuffer (0.01 M Hepes/0.0074 M KOH/0.1 M KC1, pH 8.0) toremove ammonium sulfate. The protein concentration wasdetermined spectrophotometrically by using e280 p.i = 1.32ml/mg-cm (12). Where necessary, the enzyme was concen-trated by using Amicon Centricon 10 centrifugal ultrafiltra-tion devices. PGI activity was typically 580 units/mg whenassayed spectrophotometrically in 0.1 M Hepes/0.1 M KC1,pH 8/8 mM F6P/10 units of G6P dehydrogenase (G6PDH)/0.5 mM NADP, 25°C.G6P, F6P, and M6P were obtained as their sodium salts

from Sigma. G6P was assayed spectrophotometrically (340nm) with NADP (0.3 mM) and G6PDH (bakers' yeast, Sigma)in a buffer containing 0.1 M triethanolamine, 0.1 M KCl, 0.01M MgCl2 (pH 7.6). F6P and M6P were assayed in the samecuvette by sequentially adding PGI and then phosphoman-nose isomerase (PMI, yeast, Sigma), recording the A30endpoint after each addition.

Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phos-phate; M6P, mannose 6-phosphate; PGI, phosphoglucose isomerase;PMI, phosphomannose isomerase; G6PDH, G6P dehydrogenase;kat, catalytic rate constant.*To whom reprint requests should be addressed at: Institute forCancer Research, Fox Chase Cancer Center, 7701 Burholme Av-enue, Philadelphia, PA 19111.

1237

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 90 (1993)

P-G6P

105 100 95 90 85 80 75 70ppm

FIG. 1. 13C NMR spectra showing PGI-catalyzed equilibriaamong [2-13CIG6P, [2-13CIF6P, and [2-13C]M6P. Conditions were asfollows: 10 jAmol of [2-13C]G6P, 50 mM Hepes, 0.1 M KCI (pH 8.0)in 0.5 ml of5% 2H20 at 25C (A). Spectra were obtained 3 hr (B) and4 days (C) after adding 5 nmol of PG1 (25 ul); spectra are offset fromone another.

[2-13C]G6P was enzymatically synthesized from [2-13C, 99atom %]glucose (MSD Isotopes) by incubating 0.5 mmol of

13C]glU[2- cose with 0.55 mmol of phosphoenolpyruvate-Na3,50 gmol of ATP-Na4, 0.5 mmol of KCI, O- 1 MMOI OfM902 ina 10-ml vol of H20, pH -.:-7.2. Rabbit muscle pyruvate kinaseand yeast hexokinase (20 units each from Boehringer Mann-heim) were added. After complete glucose phosphorylation

1238 Biochemistry: Seeholzer

was achieved (monitored by G6PDH/NADP assay of acid-quenched aliquots), the reaction mixture was acidified withtrichloroacetic acid and centrifuged to remove precipitatedprotein; the trichloroacetic acid was then extracted threetimes with 3 vol of ether. G6P was isolated by chromatog-raphy on Dowex-l-chloride, eluting with 20 mM HCL Frac-tions containing G6P were combined and neutralized withNaOH; the volume was then reduced by flash evaporation.Purity and identity were verified by 13C, 31p, and IH NMRspectroscopy. 1H NMR spectra were as expected for[2-13C]G6P, except for contaminating singlet resonances at5.36 and 5.19 ppm and an additional singlet at 1.92 ppmarising from acetate (degradation product of pyruvate). 31pspectra revealed a single major peak at --5 pprn (G6P) and aminor peak at --A07 ppm (-%2% of total integral) assigned tophosphoenolpyruvate. 13C spectra revealed only two reso-nances, corresponding to the a andB anomers of [2-13CIG6P.[2-2H]M6P was made by dissolving 0.5 mmol of M6P

(Sigma) in2H20foflowed by flash evaporation and redisso-lution in2H20. This step was repeated until the calculated2H20 enrichment of the water solvent exceeded 990%. Ap-proximately 10 units of PMI in 2H20 [prepared by dialyzingthe yeast enzyme against several changes of 0.01 M Hepes/0-001 M M902 (pH 7.5) in2H201 and 10 ILMOI Of M902 in2H20were added, and the H2-M6P exchange with themedium (5ml of 2H20)was followed by IH NMR spectros-copy. After the formation of [2-2H]M6P by exchange wascomplete, the medium was acidified With trichloroacetic, acidand centrifuged to remove precipitated protein; the trichlo-roacetic acid was then extracted three times with 3 vol ofether. [2-2H]M6P plus[J_2H]F6P was isolated by chromatog-raphy on Dowex-1-chloride and neutralized as describedabove, giving 2 umol of G6P (contaminant of M6P startingmaterial), 215 jurnol of [1-2H]F6P, and 263 Amol of[2-2HIM6P. G6P and [J_2H]F6P were quantitatively con-verted to 6-phosphogluconate by adding 1 jAmol of NADP,::w18 units of G6PDH, and 10 units of PGI in a 10-ml vol.NADP was regenerated by adding 450 limol of oxidizedglutathione and =10 units of glutathione reductase' (Boeh-ringer). Progress of the NADP reduction and subsequent

C G6P, D20 + PGI F M6P, D20 + PGI

5.0 43 ppm 4.0 3.5 5.0 43 ppm 4.0 3.5

FIG. 2. 1H NMR spectra showing PGI-catalyzed equilibrium among F6P, G6P, and M6P. Conditions were as follows: 10 jAmol of startingsubstrate (G6P in A, B, and C; or M6P in D, E, and F), 50mM methyfin'tidazole, 0. 1M KCI in 0.5 MI Of2H20 (p2H 8.0) at 25C. NMR acquisitionwas as follows: 128 scans, 10-sec relaxation delay, spectrometer frequency 300.14 MHz (1H), spectral width 3333.33 Hz, 8000 data points. Afterinitial spectra (A and D) were collected, 3.6 nmol of PGI (20 ILI) was added to the NMR tubes, and spectra were collected 0.5 hr later (B andE). Spectra were also collected 10 (C) and 11 (F) days after PG1 addition.

F6P G6P MR, at equilibrium F6P G6P MR, at equilibrium

'\Ii

B G6P, D20 + PGI E MR, D20 + PGIF6P G6P, at Nuihbdwn

'L - --i ik-1111-A G6P, D20 HI-aI D M6P, D20

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Proc. Natl. Acad. Sci. USA 90 (1993) 1239

Table L Equilibria among F6P, G6P, and M6P catalyzed by PGI

Integral, Ratio (literature value)% of total a,8 X6P/Y6P

a-G6P 21.9 0.58 (0.59*)/3-G6P 37.7

,8-F6P 15.6 0.26 (0.23t)a-M6P 12.8a-M6P 12.8 1.62 (1.6t)RatioG6P/F6P 3.0 (3.3§)M6P/F6P 1.1 (1.11)G6P/M6P 2.9 (3.011)Sources of data are indicated in footnotes:

*Plesser et al. (13); pH 7.6 at 250C.tKoerner et al. (14); 0.7 M, p2H 8.6 at 350C.tFrom anomeric proton resonances (Fig. 1D).§Dysan and Noltmann (15); pH 8.5 at 30'C.$Gracy and Noltmann (16); pH 7.5 at 30'C.'lCalculated (G6P/F6P)/(M6P/F6P).

reoxidation was monitored spectrophotometrically at 340 nm.Upon completion [2-2H]M6P was separated from the 6-phos-phogluconate and other compounds by chromatography onDowex-1-chloride as described above. Purity of M6P wasestablished by three criteria: (i) 1HNMR spectra of [2-2H]M6Pin 2H20 revealed no other resonances except those expectedfor this compound. (ii) [1-14C]G6P (New England Nuclear),added to the sample containing the mixture of G6P and[1-2HJF6P and [2-2H]M6P, was completely converted to6-phospho[1-14C]gluconate by G6PDH and retained by theDowex-1-chloride column (i.e., no 14C counts appeared in thefinal [2-2H]M6P solution). (iii) Enzymatic assay of [2-2H]M6Pfor G6P, F6P, and 6-phosphogluconate (assayed by NADPplus 6-phosphogluconate dehydrogenase) revealed negligiblequantities (

Proc. Natl. Acad. Sci. USA 90 (1993)

0.045

[2-'H]M6P0.04 -

0.035 -/ ~~~~~~[2_2H]M6P

0.03 - 600

U

.y0.025 4

0.02

0.015--4 -2 0 2 4 6

1/[M6P] (1/mM)0.01 -

0.005 -[2-2H]M6P

0p

0 1 2 3[M6P] (mM)

FIG. 3. Aldol C2-epimerase steady-state kineticswere measured in the M6P -+ G6P/F6P direction wphotometric assay coupled to NADP reduction with Itions were as follows: 0.1 M Hepes (pH 8) at 30'C, 0.M MgCl2, 0.3 mM NADP. (Inset) Data in double-re(Curves and lines are derived from results of nonlinleast-squares regression analysis. v, Velocity; E, enz

ruled out by the observation that in 2H20 and th(PGI, the anomeric protons of both M6P andexchange with deuterons from the medium, evenin the presence ofactive enzyme. These long-ternpresented here, but the enzyme activity wasobserving transfer of saturation (both 13C anibetween various rapidly interconverting species (

anomer pairs and between a-G6P and a- and f-F6P). The twoisomerase equilibria catalyzed by PGI and PMI are connectedby F6P, and both isomerases catalyze rapid exchange of theactivated proton oftheir substrate with the solvent (3, 5). Roseand O'Connell (2) established the absolute stereochemistry ofthe proton abstracted from F6P (pro-R) by PGI, and it wasalready known that PMI uses the other (i.e., pro-S) protonfrom that used by PGI (5). The pro-R and pro-S protons ofF6Pbecome the anomeric protons ofM6P and G6P, respectively,when these reactions are catalyzed by PMI and PGI, respec-tively. The observed stability of the anomeric protons to

46P exchange rules out contaminating PMI based on the followingrationale. Indeed, when PMI and PGI are added together tosamples like those for which spectra are shown in Fig. 2, the

8 10 G6P/M6P equilibrium is rapidly achieved with a concomitantloss of all anomeric proton resonances from exchange with the2H20 medium.The previous finding that PGI catalyzes the exchange of the

activated proton ofits substrate with the medium is also shownin Fig. 2. Thus, upon addition ofPGI, the H2 fBresonance (Fig.

4 2A) disappears (Fig. 2B and D) with a concomitant loss of spincoupling to H1-P which changes from a doublet to a singletresonance. A similar loss of coupling is seen in the Hi-a

of PGI. Rates resonance as well as the H3-a resonance, which changes fromith a spectro- a strongly coupled triplet (Fig. 2A) to a doublet (Fig. 2 B andG6PD. Condi- C) upon PGI addition. The anomeric protons of M6P (Hl-a,1 M KC1, 0.01 Hi-f3) appear as singlets in Fig. 2D because their spin couplingciprocal form. to a H2 proton in the equatorial position (i.e., in a- andear and linear 3mannopyranose-6-phosphate) is much less than the corre-!yme. sponding spin coupling in a- and (3-glucopyranose-6-

phosphate. This observation is explained, in part, by thepresence of Karplus equation, which gives coupling constants as a func-G6P do not tion of dihedral angle. The singlet H1 resonances ofM6P mayafter months also be a consequence of rapid conformational averagingn data are not among various chair- and boat-conformations of M6P.verified by A single mechanism (Scheme I) is proposed to relate the

d 1H NMR) present findings of aldol C2-epimerase activity to the previ-'i.e., between ously known isomerase and anomerase activities. The

CH20P0

HWH ~C3-24H OH

OHG6P

ftlrr~

CH20P

HHOHD

HO OH

H OHa-G6P

CH20P

HVH

H Da-M6P

CH20P

HOH D

D

0 CH20POC

\L-H (A') HHO D HO Hj11-F6P

(D)

H H 1i D/~~~.z::-H B- ID

C3-2 C3 2 C3-2 O10H \\ (A')D OH 0

c l(re-fwe)

(D) 1FoL-

D OH\ OH Ii- 1OC3A2 CA-2,\)°° - (B)- \1-OHH H

H

(A)_-lC3-2

HO}

0(A)_C3-2,

HO}

CH20P O

H

HO H5-F6P

(A) Hemiacetal ring cleavage/formation(A') Hemiketal ring cleavage/formation(B) Proton abstraction/addition(C) Rotation about C1-C2 bond axis I f(D) Rotation about C2-C3 bond axis I

Scheme I

1240 Biochemistry: Seeholzer

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Proc. Natl. Acad. Sci. USA 90 (1993) 1241

scheme considers the various enzyme-bound species thatmight exist in a completely equilibrated mixture of F6P, G6P,and M6P. The activatible substrate proton is represented asD(2H). Proton-transfer reactions are drawn horizontally;transitions between hypothetical rotamer populations aredrawn vertically. Mutarotation of the aldopyranoses (G6Pand M6P) is catalyzed by first cleaving the hemiacetal (re-action A) and then allowing rotation about the C1-C2 bondaxis (reaction C) followed by reformation of the hemiacetal tothe opposite face of the carbonyl. Mutarotation of the keto-furanose (F6P) is catalyzed in much the same way, excepthere a rotation about the C2-C3 bond axis is required toexpose alternate faces of the carbonyl to attack by the C5hydroxyl to reform the hemiketal. As mentioned in theintroduction, mutarotation of the aldopyranose and ketofura-nose substrates has clear experimental support (7, 9-11, 13).The C2-C3 bond rotation requires a larger volume than doesthe C1-C2 bond rotation. This rotation may be accommo-dated by a large enough static active site or, since the activesite resides in a cleft between two domains (17, 18), byinterdomain librational motions. Isomerization in Scheme Iproceeds by proton abstraction (reaction B) from C2 of thealdose or from C1 of the ketose to form the cis-enediolintermediate. Reprotonation at C1 or C2 from the re-face ofthe enediol completes the isomerization. Epimerization oc-curs when the cis-enediol rotates about the C2-C3 bondaxis, thus exposing the si-face to the acid conjugate of thecatalytic base. Reprotonation at C2 yields M6P. In fact,reprotonation of C2 from the si face is difficult, as evidencedby the large isotope effect (Tables 2 and 3) and by the -2 x105-fold lower rate of M6P formation relative to the isomer-ization rate of F6P. Because the anomeric proton of G6P isstable to exchange with the medium, reprotonation at C1from the si face does not occur, which may suggest someconstraints on the active-site geometry in PGI.A trans-enediol mechanism (Scheme II, where D = 2H)

Da vOH

0-HI

[2_2H]M6P

0

I //°

OH HO D

tranedol El-pro-R-2H]F6P

Scheme H

could equally well account for the C2-epimerization datapresented here. In this model, the interconversion ofG6P andF6P proceeds through the cis-enediol intermediate as de-picted in Scheme I, whereas the formation of M6P proceedsvia a trans-enediol formed by abstracting the pro-R protonfrom F6P. In this case, no trans-enediol flip is required. Thecis-enediol flip model of Scheme I appears more favorablethan the trans-enediol model (Scheme II) because of the clearrelationship it shows among mutarotase, isomerase, andepimerase activities in PGI.The possibility of a two-base mechanism for epimerization

was considered. Results ofexperiments to demonstrate intra-molecular transfer of 3H from [2-3H]M6P to [2-3HJG6P (trap-ping the latter with G6PDH/NADP) were inconclusive. Thehigh primary kinetic isotope effect (3H/H = 18.6) makesconversion of [2-3H]M6P very slow (=0.002 sec'), whereasthe rate of 3H exchange with the medium remains high (-350sec' compared with the catalytic rate constant kcat for G6Pisomerization of -700 sec1). For proton transfer to and fromM6P in the rate-limiting step of the epimerase reaction, thecis-enediol need only flip at a rate > -0.4 sec1, leavingample time for 3H exchange with the medium. Although thefinding of little or no transfer of 3H to 6-phosphogluconate isstill consistent with the mechanism proposed in Scheme I, a

two-base mechanism cannot be ruled out at this time. A morehighly refined x-ray crystallographic structure would beexpected to shed some light on this issue.

Is this aldol C2-epimerase activity a general feature ofketolisomerases? For triosephosphate isomerase, D-glyceralde-hyde-3-phosphate, the physiological enantiomer, would beproduced from L-glyceraldehyde-3-phosphate. Indeed, Rich-ard (19) has characterized this enzymatic reaction and findsthat kct is some 106-fold lower than that for isomerization,very similar to the findings on PGI presented here. Deter-mination of the stereochemistry of proton incorporation intoC3 of dihydroxyacetone phosphate will be necessary for adetailed discussion of possible reaction mechanisms for thetriosephosphate isomerase-catalyzed racemization of glycer-aldehyde-3-phosphate. Some experiments like those re-ported here with rabbit muscle PGI have been done withyeast PGI (Sigma). Similar ratios of ketol isomerase to aldolC2-epimerase were found for both enzymes. PMI was foundto have no detectable aldol C2-epimerase activity by themethods used here. This finding is consistent with the inabil-ity of PMI to mutarotate the M6P or G6P anomers. It is notyet known whether PMI can mutarotate F6P anomers, but itslack of aldol C2-epimerase activity might suggest that it willnot. Hence, the aldol C2-epimerase activity of a ketolisomerase may be related to the ability of that isomerase toform an enediol intermediate together with its ability tomutarotate the anomeric forms of its substrates.

I acknowledge the generous support of Dr. Irwin A. Rose and theuse of the Bruker AM300 spectrometer of this Institute for enablingthis work. This work has had financial support from NationalInstitutes of Health Grants GM-20940 to Dr. I. A. Rose, CA-06927and RR-05539 to this Institute, and also from an appropriation of theCommonwealth of Pennsylvania.

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3086-3092.4. Rose, I. A. (1962) Brookhaven Symp. Biol. 15, 293-309.5. Topper, Y. J. (1957) J. Biol. Chem. 225, 419-425.6. Richard, J. P. (1987) in Enzyme Mechanisms, eds. Page, M. I.

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(1973) J. Biol. Chem. 248, 2219-2224.9. Balaban, R. S. & Ferretti, J. A. (1983) Proc. Natl. Acad. Sci.

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aisse-Lagae, F. & Malaisse, W. J. (1992) J. Biol. Chem. 267,210-217.

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13. Plesser, T., Wurster, B. & Hess, B. (1979) Eur. J. Biochem. 98,93-98.

14. Koerner, T. A. W., Jr., Cary, L. W., Bhacca, N. S. & You-nathan, E. S. (1973) Biochem. Biophys. Res. Commun. 51,543-550.

15. Dysan, J. E. D. & Noltmann, E. A. (1968) J. Biol. Chem. 243,1401-1414.

16. Gracy, R. W. & Noltmann, E. A. (1968) J. Biol. Chem. 243,3161-3168.

17. Shaw, P. J. & Muirhead, H. (1976) FEBS Lett. 65, 50-55.18. Achari, A., Marshall, S. E., Muirhead, H., Palmier, R. H. &

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