Affinity Labeling and Characterization of the Active ... - Semantic Scholar

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David R. Gibson and Robert W. GracyS. From the Department of Biochemistry, North Texas State University, and the Texas College of Osteopathic Medicine,.
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 255,No. 19, Issue of October 10, pp. 9369-9374, 1980 Printed in U.S A .

Affinity Labeling and Characterizationof the Active Site Histidine of Glucosephosphate Isomerase SEQUENCE HOMOLOGY WITH TRIOSEPHOSPHATE ISOMERASE* (Received for publication, March 13, 1980)

David R. Gibson and Robert W. GracyS From the Department of Biochemistry, North Texas State University, and the Texas College of Osteopathic Medicine, Denton, Texas 76203

Fred C. Hartman From the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 38730

N-Bromoacetylethanolaminephosphate was found to zymes triosephosphate isomerase (EC 5.3.1.1) and glucoseact as a specific affinity label for the active center of phosphate isomerase (EC 5.3.1.9). For example, triosephosglucosephosphate isomerase. The inactivation process phate isomerase has been termed the “perfect catalyst” (1) followed pseudo-first order kinetics, was irreversible, and detailed studies(2) have delineated theoverall structure and exhibited rate saturation kinetics with minimal of the enzyme at high resolution. While a variety of investihalf-lives of inactivation of 4.5 and 6.3 min for the gations have suggested mechanisms for the isomerization of enzyme isolated from human placenta and rabbitmus- glucosephosphate isomerase, structural-functional studies cle, respectively. The pH dependence of the inactivation have been notably lacking. For example, although it hasbeen process closely paralleled the pH dependence of the well documented that the isomerization proceeds through a overall catalytic process with pK, values at pH 6.4 and cis-enediol intermediate (3, 4 ) , very little primary structural 9.0. The stoichiometry of labeling of either enzyme, as information is available concerning the active center or other determined with N-br~mo[~~C~]acetylethanolamine portions of the enzyme. phosphate, was 1 eq of the affinity label/subunit of Based on a variety of studies, several residues have been enzyme. After acid hydrolysis and amino acid analysis postulated to be involvedin thecatalytic process. These of the radioactive affinity-labeled human enzyme, only (5),lysine (5,6),glutamate (7), and arginine include histidine radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive (8). However, all of these studies have been of an indirect derivative obtained was 1-carboxymethylhistidine. Ac- nature, and several attempts to identify essential regions of affinity labeling have been, a t tive site tryptic peptides were isolated by solvent ex- the catalytic center directly by traction, thin layer peptide fingerprinting, and ion ex- best, only partially successful. The epoxide substrate analog, was utilized by O’Connell change chromatography before and after removal of 2-anhydromannitol-6-phosphate, the phosphate from the active site peptide. Amino acid and Rose(7). Unfortunately, amino acid analysis couldnot be analysis of the labeled peptides from the two species used to identify the labeled residue, because of the instability were very similar. Using high sensitivity methods for of the epoxide-modified residue toward both acid and base sequence analysis, the primary structure of the active hydrolysis. O’Connell and Rose tentatively identified the la* site was established as Val-Leu-His-Ala-Glu-Asn-Val-beled residue as a glutamate by isolating a radioactive dipepAsp(Gly,Thr,Ser)Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr- tide from exhaustive enzymatic cleavageswithpepsin and Phe. Apparent sequence homology between the cata- subtilisin (7). Schnackerz and Noltmann (6) observed that lytic center of glucosephosphate isomerase and triose- pyridoxal 5-phosphate, in conjunction with sodium borohyphosphate isomerase suggest that the two enzymes dride reduction, could label a lysine residue of the enzyme. The peptidelabeled with pyridoxal phosphate hasbeen studmay have evolved from a common ancestral gene. ied recently by Noltmann and co-workers and exhibited a sequence of Leu-Gly-(PLP-Lys)Gln (9). The present study describes an affinity label which reacts The aldose-ketose isomerases havebeen of particular inter- rapidly, specifically, and stoichiometrically with the catalytic est because of theiruniquecatalytic efficiency andtheir center of the enzyme. The study has allowed primary structure absolute stereospecificity. Most of the structural-functional analysis of the region surrounding an active sitehistidine.’ mechanistic studies have centered on the two glycolytic enRESULTS

* This work was supported in part by research grants from the National Institutes of Health (AM14638, AG01274) and the R. A. Welch Foundation (B-502).Part of this work was submitted by D. R. G . as partial fulfillment for the requirements for doctor of philosophy at North Texas State University. Research of F. C. H. was sponsored by the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed.

*

Inactivation by N-Bromoacetylethanolamine PhosphateIncubation of human glucosephosphate isomerase with Br-

’ Portions of this paper (including “Experimental Procedures,” Figs. 1 and 2, and Tables I to 111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document 80M-410, cite authors, and include a check or money order for $1.95 per set of photocopies. Full-sized photocopies are also included in the m i c r o f h edition of the Journal that is available from Waverly Press.

9369

9370

Actiue Site Sequence of Glucosephosphate Isomerase

AcNHEtOP' resulted in an inactivation whichfollowed pseudo-first order kinetics with a limiting half-life value of only 4% min. The inactivator concentration required for halfwas 0.056mM, a value maximal rates of inactivation (&,) which compares favorably to the X,,, values of 0.12 m and 0.07 mM for glucose 6-phosphate and fructose &phosphate, respectively (10). The isomerase was protected from inactivation by substrates or competitive inhibitors. For example, when the enzyme and BrAcNHEtOP were incubated with varying concentrations of 6-phosphogluconate, half-life of inactivation values were increased from 18 to 94 min. When inactivation half-lives of the protected enzyme were plotted versus the reciprocal of the inactivator concentration limiting half-life valuesof 4.5 min were also obtained. Further evidence for competition of BrAcNHEtOP and 6-phosphogluconatefor the active site was obtained by measuring inactivation halftimes with constant BrAcNHEtOP and varying 6-phosphogluconate concentrations (Fig. 1).The apparent dissociation constant for 6-phosphogluconate (Kd)was calculated from replots of T versus the concentration of 6-phosphogluconate (e.g.Fig. 1B). These studies yielded an average Kd value of 0.09 mM, which compares favorably with the K, of the inhibitor of 0.063 mM (11).Meloche ( 12) has pointed out that such observations are strongly indicative of a specific, active site-directed probe and competition of the two compounds for the same site. Similar results were obtained when the enzyme from rabbit muscle was exposedto the reagent (e.g. a limiting half-life of inactivation of 6.3 min was obtained). The mechanism of inactivation was examinedfurther using [2-"C]BrAcNHEtOP. When the enzyme was incubated with varying concentrations of the inactivator, the incorporation of only 1 eq of reagent/subunit of isomerase was observed for either species (Table I).The stoichiometry for the inactivation is in excellent agreement with the known relationship of one catalytic center per subunit (8,13) and indicates the specificity of the reagent for a residue in the active center. Identification of the Labeled Residue-The radioactive label was not released by treatment with 1 M hydroxylamine in 8 M urea (25°C for 24 h), followed by exhaustive dialysis, suggesting that the reagent was not bound to the enzyme via an ester linkage. Following acid hydrolysis of the radioactively labeled human enzyme, the hydrolysate was subjected to amino acid analysis. Acid hydrolysis results in the formation of a radioactive carboxymethyl derivative of the residue labeledwith the reagent. Fractions werecollected as they emerged from the amino acid analyzer and analyzed for radioactivity. A single radioactive derivative eluted at 43 min between alanine and valine. This elution position coincided with the elution position of authentic 3-carboxymethyl histidine (Fig. 2). No other radioactive peaks were observedin the elution profile. When the enzyme isolated from rabbit muscle was subjected to the same procedure all of the radioactivity emerged at 30 min coincident with 1-carboxymethylhistidine (data not shown). The elution positions for authentic 1- and 3-carboxymethylhistidine were in good agreement with those of previous studies (14). Structural Studies-Having established that the residue modified by BrAcNHEtOP was a histidine, studies were designed to provide a more detailed description of the nature of the active site. The details of the structural analyses are found in the miniprint supplemental section. Parallel studies were conducted on both the human and rabbit enzymes. After reaction with the affinity label, the enzyme was S-carboxymethylated, e-aminocarbamylated, and cleaved at arginine with trypsin. Thin layer peptide fingerprints revealed a single The abbreviation used is: BrAcNHEtOP, N-bromoacetylethanolamine phosphate.

100

7'

60' 40.

20 /

10

30

~ P G mM FIG. 1. 6-Phosphogluconateprotection of glucosephosphate isomerase from BrAcNHEtOP phosphate inactivation. A, incubations were at 37°C in 50 mM triethanolamine/HCl, pH 8.0, glucosephosphate isomerase concentrations were 5 units/ml and BrAcNHEtOP was 0.1 m ~The . following concentrations of 6-phosphogluconate were included: (e)10 mM;).( 5 mM; (A)2.5 mM; (0) 1.25 mM; (A) none. The rate of inactivation in the absence of 6phosphogluconate (A) is based on three additional data points (not shown) which are below 10%remaining activity. B , inactivation halflife as a function of the 6-phosphogluconate (6PG)concentration.

TIME

/MINI

TABLE I Stoichiometry of reaction of N -bromoacetylethanolamine phosphate to phosphoglucose isomerase Enzyme

Human Human Human Rabbit

Ratio of inactivator to enzyme

Percent inactivation

20:1

80

1.30

40: 1

89 93 90

0.80 1.10

60:l 40:I

Ratio" of label to enzyme'

1.10

Temperature 37"C,pH 8.2,50 xnh~triethanolamine HCI with 0.1% 2-mercaptoethano1. Enzyme concentration was 1 mg/ml. Incubation was 45 min, followed by gel filtration. Calculated on the basis of equivalents incorporated per subunit (M, = 65,000) of inactivated isomerase.

$

0

0 d W

u

4 IT

51

i 30

20

40 TlME

50

60

70

80

(MINUTES)

FIG. 2. Amino acidanalysis of humanglucosephosphate isomerase labeled with BrAcNHEtOP. The enzyme was reacted with the active site reagent (1:20 mole ratio) until 90% inactivation occurred. The enzyme was passed through a Sephadex G-25 column to remove excess reagent, anddialyzed exhaustively. Amino acid analysis was performedafter 24-h acid hydrolysis at 100OC. The solid line represents a tracing of the fluorescence detection of amino acids and the dashed line represents radioactivity. The two arrows show the elution points for authentic 1-carboxymethyland 3-carboxymethyl histidines.

radioactive peptide which was recovered and subjected to amino acid analysis.The composition of the active site peptide from both species showed a high degree of homology. The active site peptide from rabbit muscle wasisolated by solvent extraction and ion exchangechromatography before and after

Active Site Sequence of Glucosephosphate Isomerase enzymatic removal of the phosphate group from the affnitylabeled peptide. Edman degradation of the isolated peptide was found to proceed routinely until reaching the labeled histidine residue at which point further degradation was blocked. The sequence of the peptide was thus established by enzymatic and chemical cleavage of the primary peptide and subsequent analyses of the secondary peptides. Details of the structuralanalysis are presented in the miniprint supplement. From a combination of NH2- and COOH-terminal enzymatic degradations, Edman degradation, and overlap information from the enzymatic and chemical degradations, the following sequence was established for the active site peptide

H He,$-H2C

9371

-

*:

from the enzyme from the rabbit muscle: Val-Leu-His-Ala-

Glu-Asn-Val-Asp-(Gly,Thr,Ser)-Glu-Ile-(Thr,Gly,His,Lys,G1x)Tyr-Phe. By comparing the sequence from the rabbit peptide with the composition of the humanpeptide and assuming sequence homology, the following peptide would be predicted for the human active site region. * Val-Leu-His-Ala-Glu-Asn-Val-Asp-(Gly,Thr,Ser)-GluIle-(Gly,Thr,Hie,Lys,Glx)-Tyr-Phe-(Ser,Glx,Gly~)-Arg DISCUSSION

BrAcNHEtOP satisfies all of the criteria for a specific affhity label of the catalytic center. The reagent causes a complete, rapid inactivation, obeys saturation kinetics, covalentlyreacts stoichiometrically with the enzyme, and the inactivation can be inhibited by the presence of competitive inhibitors and substrates, The human enzyme was alkylated specifically at position 3 on the histidine imidazole ring, while the rabbit enzyme was alkylated at position N-1. Thus, the active site labeling of each form was characterized by an isomeric specificity in alkylation. The solvent extraction and bacterial alkaline phosphatase techniques greatly facilitated the isolation of the active site peptide. However, once the peptide was isolated, structural analysis was hindered by the abrupt halt in Edman degradation encountered on the thirdcycle. A possible mechanism by which this blockage may occur is shown in Fig. 3. The attack of the sulfur atom might take place at the amide linkage of the derivatized imidazole rather than at the peptide backbone amide bond. A similar, abrupt blockage of Edman degradation has been known to occur with some glycoproteins. Shively (15) found that when subjecting carcinoembryonic antigen peptidefragmentsto Edman degradation, the degradation would proceed routinely until a glycosylated asparagine became the NHZ-terminal residue, after which the degradation

n FIG. 3. Possible mechanism for blockage of Edman degradation by BrAcNHEtOP labeling. After reaction of the peptide with phenylisothiocyanate the cyclization step occurs by nucleophilic attack of the sulfur on the carbonyl of the derivitized imidazole rather than on the peptide carbonyl. v i n d i c a t e s the remainder of the polypeptide.

it is possible that the deletion of the His3 could lead to the structure found in the active site region of triosephosphate isomerase. Deletion of Tyrs could have led to the structure found in the active site of glucosephosphate isomerase. The amino acid designated as X in the ancestral protein is unclear. A single nucleotide base change can not allow substitution of asparagine for proline or vice uersa. Thus, at least two point mutations would be required to result in asparagine in this position in the glucosephosphate isomerase and proline in the triosephosphate isomerase. On the other hand, another residue such as histidine (CAU or CAC) could have been in position X. A single point mutation could result in the substitution by asparagine (AAU or AAC)in the evolution of glucosephosphate isomerase while another point mutation could result in the substitution by proline (CCU, CCC) in the case of the triosephosphate isomerase. X-ray crystallographic studies of swine glucosephosphate isomerase (8) suggest the active site nucleophile is at or near a sharp bend, and neara conformational transition point between an a helix section and a region of fl sheet. Utilizing Chau and Fasman conformational prediction methods (20), the fiist 5 residues of the active site peptide would be given an average a helix tendency () of = 1.24, but an average fl sheet tendency () of only = 0.96.

*

Val-Leu-His-Ala-Glu-Asn-Val-Asp(Gly,Thr,Ser)-Clu-Ile-(Thr,Gly,His,Lys,Glx)-Tyr-Phe a helix # turn I or /3 sheet /3 sheet could not be continued. Fortunately, leucine aminopeptidase degradation of the peptide was not so limited by the modification. Comparison of the sequence of the active site peptide of glucosephosphate isomerase (GPI) to the sequence surrounding the active site nucleophile in triosephosphate isomerase (TPI) (16-19), reveals a degree of sequence homology. * GPI -Val-Leu-His-Ala-Glu-Asn-Val * TPI -Val-Leu-Ala-Tyr-Glu-Pro-Val

If a common ancestral protein had thefollowing sequence Val-Leu-His-Ala-Tyr-Glu-X-Val 1

2

3

4

5

6

7

8

Therefore, a helixis strongly indicated. The Asn-Val-Asp(Gly,Thr,Ser) region is predicted as p turn, but with the identity of the 9th residue in doubt (Gly, Thr, or Ser), anda of 0.86 uersus a of 0.90, the only structure which is disallowed is a helix. The third region is predicted as fl sheet because > (0.91 > 0.82), but since is not greater than 1.0, this assignment is tentative. The histidyl residue that is alkylated selectively by BrAcNHEtOP could be the acid-base group that effects aldehyde-ketone interconversion. However, the glutamyl y-carboxylate, only 2 residues distal in sequence from the labeled histidyl in the isolated active site peptide, could also serve such a function analogous to the triosephosphate isomerase reaction. Establishment of the identity of the active site

9372

Site Active

Sequence of Glucosephosphate Isomerase

residue labeled by 1,2-anhydro-~-mannitol (7) should help in making this distinction. REFERENCES 1. Knowles, J. R., and Albery, W. J. (1977) Accts. Chem. Res. 10, 105-110 2. Banner, D. W., Bloomer, A. C., Petsko, G . A., Phillips, D. C., Pogson, C. I., Wilson, I. A., Corran, P. H., Furth, A. J., Milman, J. D., Offord, R. E., Priddle, J. D., and Waley, S . G. (1975) Nature 255,609-614 3. Rose, I. A,, and OConnell, E. L. (1961) J . Biol. Chem. 236,30863092 4. Schray, K. J., Benkovic, S. J., Benkovic, P. A., and Rose, I. A. (1973) J. Biol. Chem. 248,2219-2224 5. Dyson, J. E. D., and Noltmann, E. A. (1968) J. Biol. Chem. 243, 1401-1414 6. Schnackerz, K. D., and Noltmann, E. A. (1971) Biochemistry 10, 4837-4843 7. O’Connell, E. L., and Rose, I. A. (1973) J. Biol. Chem. 248,22252231 8. Riordan, J. F., McElvany, K. D., and Barders, C.L., Jr. (1977)

Science 195,884-886 9. Noltman, E. A., Palmier, R. H., Gee, D. M., and Bisset, A. D. (1979) Fed. Proc. 324 10. Tilley, B. E., Cracy, R. W., and Welch, S. G. (1974) J Biol, Chem. 249,4571-4579 11. Gracy, R. W., and Tilley, B. E.(1973) Methods Enzymol. 41,392399 12. Meloche, H.P. (1967) Biochemistry 6,2273-2280 13. Bruch, P., Schnacken, K. D., and Gracy, R. W. (1976) Eur. J. Biochem. 68, 153-158 14. Gurd, F. R., (1972) Methods Enzymol. 25,4571-4579 15. Shively, J. E., Kessler, M. J., and Todd, C . W. (1978) Cancer Res. 38,2199-2208 16. Hartman, F. C. (1971) Biochemistv 10, 146-154 17. De La Mare, S., Coulson, A. F. W., Knowles, J. R., Priddle, J. D., and Offord, R. E. (1972) Biochem. J. 129,321-331 18. Norton, I. L., and Hartman, F. C. (1972) Biochemistry 11,44354441 19. Hartman, F. C., and Gracy, R. W. (1973) Biochem. Biophys. Res. Commun. 52,388-393 20. Chau, P. Y.,and Fasman, G. D. (1978) Annu. Reu. Biochem. 47. 251-276 Additional references are found on p. 9374.

Site Active

Sequence of Glucosephosphate Isomerase

9373

RESULTS

SUPPLEMENTARY WTERIAL TO AFFINITV LABELINGAN0CHARACTERIZATION OF THE ACTIVE-SITE HlSTlDlNE OF GLUCOSEPHOSPHATE ISOMRASE: SEQUENCE HOWLOGY UlTH TRIOSEPWSPHATEISOMERASE David R. Gibson. Robert U. G r l c y and Fred C. Hartman EXPERIMENTAL PROCEDURES Enz mel: B a c t e r i a la l k a l i n e pholphltale,gluc0le-6-ph(llphdte dehydrogenase, carb o w p Z F k e 8 , carboxypeptidase A. leucine m i n o p e p t i d a s e ,t r y p s i n( d i p h e n y l c a r bamyl chloride-treated).ch-trypsin, pronase.and cathepsin C were a l l obtained frm Sigma ChemlcalCmpany. 3. V8 pmteare(rtapnylococcalprotease) was from n l l e s Laboratones.

aureus

Substrater and I n h i b i t o m :F r ~ ~ t ~ l e - B - p h O l p h d t e , glucose-6-phosphate.6-phosphogluconate. and NADP+ !ere obtainedfromSigna.5-Phorphoardbingnlte vas synthesized and generouslysupplied by K.D. Schnackerz. N-Bromacetylethanalam,ne phosphate (BrAcNHEtOPI was synthesized I S d e r c r l b e dp r e v i o u s l y( 1 )f r o mb r o w a c e t i ca c i dl a b e l e d parition. i n the [I-"C] ChrMiatOgrdJhlc and Electrophoretic Supplier: C e l l u l a r e phosphateCation exchanger Tccaarre mesh) w i t h an exchange c a p a c i t y of 1.0 meqlgm was obtainedfmm S i g m Chemical Capany. Carbo-thy1 BiogelCation exchanger(153-0840) and theCation exchanger AG 50Y X-8 y e s i n (100-200 mesh; 1 . 7 m e q l m l ) were Obtained fmm BloRadLaband O l d t o r l e l . Sephadex G-50 (ruper f i n e ) was obtained frm Phamacld.Ampholiner a l lo t h e rr e a g e n t sf o ri s o e l e c t r i cf o c m l n g were f r o * LKB Instruments.Allreagents Were frm B1ORad L d b o r l t o r l e 5 . Amino fop SO8 p a l y a c r y l a m i d eg e le l e c t r o p h o r e s i r acldstandards 411 obtained f m m Beckmn lnstrumentr.,~~~ and a l l o.t h e.r reaoents f o.r ~ .~ =... amino acid a n l l y l i l were frm O i o n e x .P r e c o d t e d .p l a r t l c - b a c k e d ,t h i nl a y e rc e l l u l o s e 400-221. Analyticalgrade column^ f o r i o n w e e t i were frm Brinkman(Mchen-Nagel, were develexchange p e p t i d ei s o l a t i o n (0.6 x 50 cm) were fromBiaLab.Chrmdtogrdms oped w i t hf l u o r e r c a m i n e( I - p h e n y l i p l r o [ f u r a n - Z( 3 H ) , -l'-phthalan]-3,3'-dimel frm Hoffman LaRoche. ~

TABLE I COMPARISON OF THE HUMAN AN0 RABBlT GLUCOSEPHOSPHATE ISOMERASE ACTIVE-SITE PEPTIDES'

~~

Amlno Acld

M i r c e l l a n e a u rR q e n t i :I o d o a c e t i ca c i d( E a r t m a nC h e m c a i r ) vas r e c r y r t a l l l i e d P r l o r t o use. GuanidinimChloPide. sodium dodecylsulfate and d d n s y lc h l o r i d e( 5 were obtalned frm P i e r c e C h m i c a l r . dimethylaminonapthalene-1-rulfanylChloride) and phenyliSothi0Cyanate OABITC (4.N.N-d~meth~laminoarobenrene-4'-~rothia~yanate) (PlTC) Yere obtained flm P i e r c e ChemicalCmpany.

AIX Thr

2 0 (2) 1.8 (21 1 7 (2)

S W GIX

1 l O I d t l o no fG l u c a r e Phosphate l r m e r a ~ e : I s o l d t i o n [if glucorephorphate r~ainerli~e from hum"placentae was p e r f o d by a M d i f i c a t i m (2) o f t h e I u b s t r a t e e l u t i o n method Gvacy and T i l l e y ( 3 ) . The enzyme was hmogeneaurby a l k a l i n e and 808-polyacrylamide gel e l e c t l o p h o r e s l s andexhib,ted L p e c i f l c a c t i v i t i e s o f 8Mi-1000 i n t e r n a t r o n a l u n l t s i m g

G~Y Ala CY5 (0) Val Met 11e Le" T rY Phe

Of

I s o l a t i o no fg l u c o s e p h o 5 p h a t ei s m e r a s e from r a b b l t muscle was c a r r i e d Outaccordi n g t o P h i l l i p s e t a 1 ( 4 )w i t ht h ef o l l o w f i ge x c e p t i o n s :b u f f e r 1 was 50 mM t m e t h d n o l b u f f e r I1 vas 10 mM amine-HC1, 1 m EDTA. 0.11 (v/v) 2-mrcaptoethanol, pH 8.2,and imidazole-HC1.1 mH EDTA. 0.1% ("1") 2-mercaptoethanol. pH 7.2. The c e l l u l o r e phosp h d t e d i a l y s a t e Ma$ washed wrth buffer 11 u n t i l t h e d b m r b a n c e a t 280 MI YLI 0.05 or less, then the enzyme was e l u t e d w l t h 3 mH glucore-6-phosphate.

*Rabbst

'HUTd" Reriduer/mle Reslduer/mole

(1)

4.0 4.1 1 . 0 1.0 0.0 2 2 0.4 1.4 0.8 0.7 0.6

HI6

3

2.2 (2)

1.8 (21 1.1 ( 1 ) 2.8 (31 2.3 ( 2 )

(4)

(I) (1)

(21 (0)

(I) (11 (1) (1)

(1)

0.0 (01 1.8 (2) 0.0 (01 1.0 1.0 ( 1 ) 0.6 ( 1 )

1 2 (11

1.1 ( 1 ) 1 . 1( 1 )

0.1 ( 0 ) 0.2 ( 1 )

0.0 (01 0.3 (11

Arg

cm

E n i y m t l cD i g e s t i o n s : BrAcNHEtOP labeledglucosephosphate l~omerasewas denatured Alkvlation O f ramdies were d i a For Specificcleavage a t a r g l n l n e , peplyzedagalnrtwater and l y o p h i l i z e dt od r y n e s s . t l d e s were resuspended i n 0.5 M N-ethylnrorpholine-HC1. pH 8.5 c o n t a i n i n g 6 M guanidine h y d r o c h l o r i d e and 0.5 M EDTA and were r e a c t e d w i t h pOtass1um Cyanate t o b l o c k t h e i - d m n o groups (81. Following Carbamylation.sampler w e ~ ed i a l y z e d ,l y o p h i l i z e d and resuspended i n 0 . 2 M a m n i u n bicarbonate. pH 7.8 (250 " 1 ) . T ~ y p s l n was added i n three 2 h o u r I n t e r r a l r , and the digestions were s t i r r e d Continuously and equalaliquot$at was 1.50 a l l o w e dt op r o c e e df o r 24 hours a t 37.. The f i n a l r a t l o o f t r y p r l n t o p r o t e l n (Wwl. Leucine m n o P e p t i d a s ed l g e i t i o n s were c a r r t e d Out by rurpendlngpeptides ? n 250 y l O f 25 mM d m n i u m bicarbonate, pH 7.8 c o n t a i n i n g 2.5 dl QCl,and l n w b d t l n g t h ep e p t i d e 6a t 37. w t h l e u c i n e arnlnapeptidare 1 0 d 1.50 ( w / w ) r a t i o o f p e p t i d a s e t o protern.Aliquot3 wepe w t h d r a w n .l y o p h l l l i e d . resuspended ~n 0.2 M sodium c i t r a t e , amino acidanalyzer.Cdrboxypeptidasedlgertianr pH 2 2 , and a p p l i e dd i r e c t l yt ot h e Were conductedbysuspendingthepeptldel i n 250 "1of 50 dl N-ethylmorphollne-acetdte, pH 8.0, and I n c u b a t i n ga t 37' wlthCarboxypeptidase 8 , and/or carboxypeptidase A. Aliquot *re * i t h d r a u nf o ra n a l y s i s and ? m e d i a t e l yf r o z e n and l y o p h i l i z e d . For a l k a l i n e p h o l p h a f a l ed i g e r t 7 o n st h ed r i e dp e p t l d e s (400-600 n a n m a l e r ) were suspended i n 50 mH N-elhylnxlrpholIne-acetate. pH 8.0, and I n c u b a t e d a t 37" w t h 1.0 u n i t o f b a c t e r i a l a l were c a m l e do u t by k a l l n e phosphatase, f o r 18-20 h o u r s . C h m t r y p r l n d l g e r t i o n r ~n 50 dl anmonium bicarbonate pH 7.8 and incubating f o r rerurpendingthepeptides 12 hourswithchymotrypsin, l n a 1:50 (v/*) Patloofchhotryps;"topeptlde. Staphyconductedbyrvrpendlngpeptides i n 200 p l o f 50 mH ~ O C O C E . ? ~ p r o t e a s e d i g e s t i o nwere s phosphatebuffer. pH 7.8, and i n c u b a t i n g f o r 8 hours a t 37" * I t h staphylococcalprow/u). P a r t l a l a c i d h y d r o l y s i s was c a r n e d o u t a t 1 0 9 i n 1.0 m l o f 0.03 tease L1:20 N HC1 fa? 20 hours.

B1P

i n 5 ml O f 1 M Trir-HCL, pH 8.5. c o n t a i n i n g 8 M urea and 0.5 M EDTA. cysteines was c a r r i e dO u t as p r e v i o u r l y d e i c r i b e d byGracy ( 7 ) .A l l

I n o r d e r t o prepare s u f f i c i e n t active-site labeled enzyme f o r sequence s t u d i e s t h e r a b b i t m u ~ c l e enzyme was l a b e l e d w i t h "C reagent and p u r i f i e d I S f o l l o w s . The t o a 24 l a b e l e d enzyme was 8-carbowmethylated,c-amino-carbamylated,andIubJected h o u rt r y p t i ch y d r o l y r l r . The r e s u l t i n gp e p t i d e s Were found t o be l n r o l u b l e ~n a number O f s o l v e n t systems; h w e v e r . when t h e i n s o l u b l e m a t e r i a l was e x t r a c t e d w i t h 0.2 M p y n d i o e - a c e t a t e , pH 3.0. t h er a d r o a c t i v ep e p t l d e vas s o l u b i l i z e d . T h i s m a t e n a l was d r l e d UndeP a streamofnitrogen and a p p l i e d t o a Bio-Rad AG 50-X8 Cation exchange peak o f r a d i o a c t i v i t y e l u t e d i n t h e "Old c o l u m and e l u t e d w i t h a pH g r a d i e n t .As i n g l e *as volurne. H a e v e r , t h l n - l a y e r f i n g e r p r i n t i n g rhowed t h a t t h e r a d i o a c t i v e p e p t i d e s t i l l contaminatedwithnom-radioactivepeptides.Therefore,thepeptides were incufrm t h e b a t e dW i t hb a c t e r i a la l k a l i n e phosphatase tohydrolyzethephosphatemoiety b r m a a c e t y l e t h a n ~ l a i " phosphate ~ labeledpeptide.This change i n t h e overall charge was e x p l o i t e d by SubJectlngthedephosphorylatedpeptlde to rechrmo ft h ep e p t i d e T h i nl a y e rf i n g e r p r i n t i n go ft h em a t e r i a l tography on the same Cation exchangecolumn. p u n f i e d by these methods revealed d rlngle fluorescarnine-positive and n i n h y d r i n - p o s i t i v e The peptlde was SubJected t or u b p e p t l d ev h r c h contained a l l O f t h e r a d l o a c t i v i t y . sequent 5tP"Ct"P.I analyses.

Amino A c l dA n a l p e r :H y d r o l y s l sf o r amino a c l d analyser was c a r r i e d o u t i n vacuo a t llOD f o r 24 hours i n 6 N HCl w i t h 0.02; 2-mercaptaethanal. Amino acids uerI f T e d by f l u o r e s c e n c e d e t e c t i o n u t l l l z i o a 0-Dhthaldialdehvde. t v.r t e i n e was d e t e m l n e d . d l 8 - c d r b o r m e t h y lc y s t e i n e .T r y p t o p h a n - w a rd e t e r m l n e da f t e rh y d m l y s l ri n 6 N HCI c a n t a l n i n g 2. ( v / v l 2-merCaptOaCetic a c l d ( 9 ) .

Sequence Analysis: Edman d e g r a a a t i o nw i t h OABITC (4-N. N-dianthylamnoalobenlene, 4 ' - 1 s o t h i o c a n a t e l was c a r n e d o u t b y a m d i f i c a t m n of the procedure O f Chdng e t a1 (12).Peptides were placed in 1-ml t h i c k - w a l lc o n i c a lv i a l s and d l r r o l v e d ? n 80 aqueous p y n d l n e (50% V / U ) and r e a c t e dw i t h 40 y l OABITC (10 n a n m o l e l u l o f p y r l d i n e were f l u s h e d w t h d r y mtrogen. sealed and incubated a i PFepared f r e l h d a i l y ] . Y l a l r 52" f o r SO minuter.After p ~ l m r y c o u p l i n g , 20 "1 phenylisothlocydndte was added, and a Second c o u p l r n gr e a c t i o n way a l l o v e dt op r o c e e da t 50' f o r 30 min.Excelsreagents andbY-prOdUCtl were P m w d by e x t r a c t i o n w i t h 0.5 ml h e p t a n e l e t h y l a c e t a t e 2:1 ( v i v ) Aftervortex mlxlng and C e n t n f u g a t l o n . t h e o r g a n i c phase was ?moved and dircd;ded. The r e r l d u e was d r i e d i n Y ~ C Y O . d i s s o l v e d i n 50 p l anhydrous t r i f l u o r a c e t l ca c l d . f l u s h e dw i t hd r y n 7 t r o s n x h e a t e d f o r 15 minuter a t 52O. Sample% were d r i e d I n vacuo and d i s s o l v e d i n 50 y l water.Butylacetate (200 " 1 ) was added, and the mlxt u r e WdP shaken v i g o r o u s l yf o l l o w e d by c e n t r i f u g a t i o n . A f t e r ~ e m v a l Of t h eb u t y l acetatelayercontainingthethlamlinone,thepeptideinthe aqueous phase was d r r e d i n vacuo and subjectedtothenextdegradationcycle. The b u t y l a c e t a t e e x t r a c t was d r i e d , and t h et h i a z o l i n o n e was c o n v e r t e dt ot h et h i a h y d a n t o i n by rerurpendlng ~n 20 y l water.40 41 O f acetic a c i d ( s a t u r a t e d w i t h HC1) and heatingfor 50 m i n u t e r a t 52'. "

20

40 TIME(MIN)

Fig. 1.

Carboxypeptidase d i g e s t i o n glucosephCsphateisomerase

Of

t h ea c t i v es i t ep e p t l d e

from r a b b l t muscle

B for30min., The p e p t i d e was i n c u b a t e dw i t h 0.1 U n l t ofcarboxypeptidase t h e n an aliquot M&S w i t h d r a m fer a w l y ~ and ~ l 0.1 u n i t o f c l ? b o x y p e p t l d a ~ e R was added. Incubations Were a t 37' i n 50 mM N - e t h y l w r p h o l i n ea c e t a t e pH 8.0 w i t h 0.1%2-mercaptoethanol. A l i q u o t r were d r i e d and a p p l i e d d i r e c t l y io t h ea i a l y z e r . S m a l r : ( 0 1 Phenylalamne. ( 0 ) t y r o s i n e ; ( A ) g l y c i n e . (A)

"

SVbtraCtive E h n degradatlon war c a r r l e d Out by the same procedure 1s above w l t h C w p l l n g w a ~for 50 minutes a t 52' w i t h p h e n y l l m t h i o c p n a t e o n l y . the exception that amino a c i d a n a l y r 1 r ( l 3 ) . A p w t l o n O f the sample was withdrawn,hydrolyedandIubJectedto

was conductedby digestion Carboxyl-tenninalAnalyrlr:Carbaw-terminalanalysis w t h c a v b o w p e p t i d * s e s .I n i t i & lh y d r o l y s i so ft h eP e p t i d ew i t hc w t a y p e p t i d a s e lA and B r e s u l t e d i n a r a p i d , s t o i c h i m e t r i c r e l e a s e Of t y r o s i n e and phenylalanine(Fig.1) The k i n e t i c s O f t h e r e l e a s e o f m l n o a c i d s a t l a e r e d concentrations O f carboxypeptiddle I n d i c a t e dt n e Sequence Of t e m i n a l r e l e a r e Of amino a c i d s was -Tyr-Phe. The presence Of a c a r b o w - t e r n i n u s p h e n y l a l a n i n e i s C o n l i P t e n t w i t h t h e p e p t i d e b e i n g t h e r e s u l t o f a cleavage byChmOtrypEin.

Site Active

9374

2,0

t

Sequence of Glucosephosphate Isomerase

p"----o

Several types of e n z p a t l c and Chemicalcleavages were I n v e s t i g a t e d as a d d l t i o n a1 means o f fragmentationofthepeptlde.Llmited pronase digestion over a range of d i gePtlOntiinel (30 minuter t o t h r e e h w m ) War found t o be much t o e T d n d m . on the basis o f i t s p e p t l d ef l n g e r p r i n t r .A t t e m p t s a t d i g e s t i o n w t h c a t h e p s i n c were, l i k e w i s e , UnluCCeiafUl. Slnce thepeptide contained "0 Cysteine,nethlonine, Or tryptophan relldues. r w t l n e chemical cleavages w e n a l s o l i m i t e d . However, Wo smallradioactive peptlde fragments wefe oetained b y S U b J e c t i n g t h e o r l g l n a l t r y p t i c a c t l v e s i t e p e p t l d e topartialacldhydrolysl~accordingtothe method Of SchUltz(17). The peptldes reivltlog from the partla1 dcld hydralynr of the rabbit active r i t e p e p t i d e were subJected t ot h l n - l a y e rp e p t i d ef l n g e r p r i n t r n g . The radioactive peptides were recovered from thef7ngerprints. and t h w r amino a c i d c m p o r i t i o n r d m shown i n Table 11 ( i n d l cated a s PA1 and PAZ).

AAdltlOnalpeptldefragnents were obtamed by secondaryenzymatic d > g e s t l o n * I t h chymotrypnn and staphylococcalprotease.Staphylococcalproteasecledve~proteins and p e p t i d e s a t t n e carboxy t e r n i n u s of glutamate and a l p a ? t d t e r e s i d u e s , u i t h a preference f a rg i u t a m d t e( 1 5 ) . After t h l n - l a y e pf i n g e r p r l n t i n g ,t h ep e p t l d e r wele recovered exandanalyzed for m d l m c t l v i t y . Three r a d i o a c t i v ep e p t i d e s were foundwhichwere t r a c t e d Pran the t h i n - l a v e r o l a t e s and subjected t o amino a c l d m d l v s * i . The comDoa& shown ~n l a b i e I1 SltionSoftherepeptide; The CampOsltiOn6 O f thepeptldeJ are C O n l l i t e n t w i t h t h e sequence deduced from the peptidase dlge6tlOn6. Edman degradations and p a r t i a l a c l d hydrolysis. l e ~ ~ l amino n d The c o m p o n t i o n of CS-4 was identical t o PA-1 exceptfor one a d d l t i o n b l e q u i v a l e n t o t Glu, I l e and Leu. Slnce amino a c i d a n a l y ~ lo~ft h ep a r e n tt r y p t l cp e p t l d ei n d i c a t e d d s l n g l e l e u c ~ n e . ~tmust representthe amino terminus. and ~ o m p d l l s ~ Wlth n (5-1 and CS-2 p l a c e st h eG l u - i l e sequence a t t h e CarODXYl terninus. The v a l i n e and l e u c i n e ~n Peptlde CS-1 would be due t o r e s i d u e s a t t h e amlno t e m l n u s .

T46LE 111 SUMMARY ALIGNMENT OF ALL SEQUENCE

WFORPATION

I n order t o deterninewhether 01 n o t t h i s a b r u p t h a l t i n d e g r a d d t l o n was due t o some u n i q u e p r o p e r t y o f t h e DABITC technlque or was inherent ~n thepeptlde.zubtracThe t i v e Edman d e g r a d a t i o nw i t hp h e n y l l i a t h l o c y a n a t e was performed on thepeptide. results o f t h es u b t m c t l v ed e g r a d a t l o n w e ~ e)denticaltothoseobtalnedWith DRBITC. Tne sequential release o f valine and leucine vas f o l l o w e d b y a C e i i d t i o n o f d e y r a d a t l o n . S m l l a ~ w S u l t were i Obtalnedby s o l l d phase sequence a n a l y i ? s

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Hartman. F.C., Suh. E., 8233-8239

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Welch, M.H. and Barker. R . (1973) J . B l o t .C h m .

T1a.1. 0. and Gpacy. R.U. ( 1 9 ~ 0 )Arch. Elochm. Rlophyr.200,431-491

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PuPdy, K..

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G P ~ C YR.Y. , and T l l l e y . 3.E.

(1973) h t h o d r ~n Enzymal.

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P h r l l > p s , T.L., Talent, 3.M. and Gracy. R.W. (1976) Blochem. BlOphyl.Acta 524-620 T i l l e y , B.E. and Gracy, R . W . and Welch, S.G. (1974) J . B i o l . Chem. 249, 4571-4579

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25, 103-120 S t a r t , G. (1972) h t h o d sE n z p o l . 3. 103-120 Matrubam, H., and Sasaki, R.M. (1959) B1ochem. Biophyr. Res. C m u n . 3 5 , 175-181 (1977) Methods Enzymol.

IO. Werner, A.M., P l a t t . 1. and Weber, K. (1972) J . 3101. Cher.. 11.

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( 1 9 7 5 ) " P r o t e i n Sequence Oetermlnatior".SprlngYerleg,

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Brauer 0. and Whlttmm-LlebOld,E.(1978) Kanigrberg, W (1972) Meth. Enzymol. 3. 326-332

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