Amino Acid Sequence of the Biotinyl Subunit from

THE JOURNAL OF B~LOGICAL CHEMISTRY Vol. 254, No. 22, ISIX of November 25, pp. 11615-11622, Printed in U.S.A.

Amino

Acid

1979

Sequence

of the Biotinyl

Subunit

from

Transcarboxylase* (Received for publication, April 23, 1979)

W. Lee Maloy,$ From

the Department

Lowell From

Botho

H. Ericsson the Department

U. Bowien,g

of Biochemistry,

and Kenneth of Biochemistry,

Gene K. Zwolinski,n Case

Western

Reserve

University

of Washington,

+ CH&OCOO= CHnCHzCOSCoA + -OOCCHXOCOO-

CH&H(COO-)COSCoA

reactions

each

Seattle,

of Medicine,

Washington

Biotinyl Subunit-COO-

Cleveland,

G. Wood Ohio

44106

98195

+ CH&OCOO5 Sg outside subunit e

The biotinyl subunit serves in the transfer of the carboxyl group between the substrate binding sites which are on the central and outside subunits. In contrast to some biotin-containing enzymes (2,3), the biotinyl subunit cannot be replaced by either free biotin or biocytin in either of the partial reactions of transcarboxylase ( 1). The subunit structure of transcarboxylase of Propionibacterium shermanii is quite complex. There are two enzymatitally active forms, an 18 S form stable at pH 6.8 and a 26 S form stable at pH 5.5 (4). Both forms are composed of three nonidentical subunits. The 12 SH central hexameric subunit is common to both forms. Three tetrameric subunits (designated 6 SE) are attached to the central subunit in the 18 S form and six in the 26 S form (4). The 6 SE subunits are each composed of a 5 S dimeric outside subunit and two 1.3 SE biotinyl subunits. The 1.3 SE biotinyl subunit has been isolated and found to occur in two forms (5,6). The one has been reported to consist of 123 residues and the other of 117 residues (6). The short form may arise due to proteolysis during the isolation. It has been shown that the NH*-terminal 42 residues of the biotinyl subunit are necessary to bind the outside subunit to the central subunit (7). As a part of our studies on the role of the biotinyl subunit in catalysis and in the assembly of subunits, it was necessary to determine the complete amino acid sequence of the biotinyl subunit. Preliminary reports’ concerning the sequence of this subunit from P. shermanii have appeared (6, 8-10). EXPERIMENTAL

(la)

(lb) biotinyl subunit + -OOCCHzCOCOO-

+ biotinyl subunit 12 SH central subunit y

School

and Harland

The “Experimental supplement.’

PROCEDURES

Procedures”

are described in the miniprint

CH&H&OSCoA RESULTS

+ biotinyl subunit-COO* This investigation was supported by Grant GM 22579 (H. G. W.) and by Grant GM 15731 (K. A. W.) from the National Institutes of Health. 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. $ Present address, Laboratory of Immunogenetics, Building 8, Room 100, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20205. 5 Present address, Institut fiir Mikrobiologie der Universitgt, 34 GGttingen, Grisebachstrasse 8, West Germany. 1 Present address, Ortho Diagnostics, Rte 202, Raritan, New Jersey 08869.

The complete amino acid sequence of the biotinyl subunit consisting of 123 residues is shown in Fig. 1. The majority of the evidence is based on analysis of the complete subunit and five polypeptides derived from it as illustrated in Fig. 2 (see ’ Some errors were made in the preliminary studies which will be considered under “Results.” *The “Experimental Procedures” are presented in a miniprint format immediately following this paper. The S with the figure numbers refers to the supplement. 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 No. 79M-778, cite authors, and include a check or money order for $1.20 per set of photocopies.

11615

Downloaded from www.jbc.org by guest, on September 18, 2011

Transcarboxylase (methylmalonyl-CoA:pyruvate carboxytransferase EC 2.1.3.1)) is a biotin-containing enzyme found in propionic acid bacteria which catalyzes the following reaction:

The overall reaction is the sum of two partial catalyzed by specific subunits (1) as follows:

University,

Kumar,

A. Walsh

The complete amino acid sequence of the biotinyl subunit from the enzyme transcarboxylase of Propionibacterium shermanii has been determined from the structures of overlapping tryptic and cyanogen bromide peptides together with sequenator analysis on the whole subunit. The subunit contains 123 amino acid residues. Eleven of nineteen residues in the region of biotin attachment, when compared to pyruvate carboxylase from avian liver (Rylatt, D. B., Keech, D. B., and Wallace, J. C. (1977) Arch. Biochem. Biophys. 183,113122), were found to be in identical positions relative to biocytin. There was less homology with acetyl-CoA carboxylase from Escherichia coli (Sutton, M. R., Fall, R. R., Nervi, A. M., Alberts, A. W., Vagelos, P. R., and Bradshaw, R. A. (1977) J. Biol. Chem. 252, 3934-3940), but in all of these biotin enzymes there was an alanylmethionyl-biocytinyl-methionine sequence. The secondary structure of the biotinyl subunit has been estimated using the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1978) Adu. Enzymol. 47,45-148) and considered in relationship to the role of the biotinyl subunit in the structure and function in transcarboxylase.

CH&H(COO~)COSCoA

K. Ganesh

11616

1

Sequence of Biotinyl

Subunit

- Tyr

- Asp - Val

- Asp

- Gly

- 'I%+ - Ile

- Leu

- Pro - Arg

- Ala

- Ala

- Gly

- Glu - Ile

- Pro - Ala

60 - Pro

Met - Lys - Leu - Lys

- Val

- Thr - Val

- Asn - Gly

10 - Thr - Ala

Val

- Asp

20 - Lys - Ser - His

- Glu - Am

- Pro - Met

- Gly

- Ala

- Pro

- Ala

- Lys - Ala

- Gly

- Glu - Gly

5

- Asp - Val

35

Phe - Gly

- Gly

- Gly

- Thr - Gly

Gly

- Gly

- Ala

- Gly

50

- Ala

- Gly

40

55

65

Leu - Ala

25

- Thr - Val

- Se+ - Lys - Ile

- Leu - Val

- Lys - Glu - Gly

- Val

- Leu - Val

Glu - Thr - Glu - Ile

95 - Am - Ala

- Pro

100 - Thr - Asp - Gly - Lys

Leu - Val

- Lys - Glu

110 - Arg - Asp - Ala

Lys - Ile

123 - Gly - COOH 20

40

80

60 I

I

I

- Val

- Gln

100 I

120 I

-7

T-16

w/r] T-6F

m

CB-86

m TS-SC

1

M

115 - Gly - Gly

1 I

ggg?////h T4A

85

- Leu - Glu - Ala

R

K

K

MM

izzl KR

K

FIG. 2. Strategy of analysis of the sequence of the biotinyl subunit. The designation of the peptides is described in Footnote 3. The hash marks indicate portions sequenced. For details, see Table II. The lower bar indicates the cleavage sites where iV, R, and K are

Met, Arg, and Lys, respectively. Footnote 3 for the method of designation of the polypeptides). Two of the polypeptides, T-1B and T-4A, were obtained by direct treatment of the enzyme briefly with trypsin (7). The arginyl-alanine bond between residues 42 and 43 apparently is exposed and, hence, is very susceptible to tryptic cleavage in the intact enzyme. A 79-residue biotinyl polypeptide, T-4A (residues 43 to 121), was obtained by tryptic cleavage of 26 S transcarboxylase at pH 5.5. The latter was separated from the remaining portion of the enzyme (see Figs. 2S, 3S, 4S, and Table I). A 50-residue polypeptide, T-1B (residues 52 to lOl), was obtained (7) by treatment of the 18 S enzyme with trypsin ’ The following procedure has been used to designate the polypeptides: T indicates it was obtained by trypsin action, CB by cyanogen bromide cleavage, and TS by trypsin treatment following succinylation. The subsequent number indicates the figure illustrating the isolation of the polypeptide and the letter indicates the pool of fractions from which the polypeptide was obtained. Thus, T-1B indicates the polypeptide obtained with trypsin as illustrated in Fig. 1S and contained in Pool B.

- Val

- Met

- Asp - Thr

amino acid sequence subunit from trans-

90 - Bet - Met

- Glu - Lys

- Gln - Gly

FIG. 1. The of the biotinyl carboxylase.

105 - Val

120 - Leu - Ile

at pH 6.5 (Fig. IS). A 24-residue polypeptide, T-6F, consisting of residues 78 to 101, resulted from treatment of polypeptide T-4A with trypsin at pH 8 (Fig. 6s). Polypeptide CB-8B (residues 91 to 123) was obtained by CNBr cleavage of the biotinyl subunit (Fig. 8s) and polypeptide TS-SC (residues 111 to 123) by cleavage of the succinylated biotinyl subunit with trypsin (Fig. 9s). Additional data will be considered in relationship to the sequence shown in Fig. 1 but will not be presented in detail. In Fig. 2 and Table II the results from the automated Edman degradations are summarized, including some tentative identifications and positions where a residue was not identified in that particular degradation. The majority of the sequence has been established by two completely independent degradations. In any case, the derivatives from the automated degradations were identified, with one exception, by more than one procedure (see Table II). Residues 1 to 47, as shown in Fig. 2, have been determined by automated Edman degradation of the biotinyl subunit. The longer degradation through residue 55 was reported in preliminary communications (6,810). Residue 24 was reported as aspartic acid, but the more recent degradation in which the residues were identified both by HPLC* and GLC4 has shown that this residue is asparagine. Methionine at residue 26 was identified in only one of the two degradations of the biotinyl subunit. However, the results from cleavage with CNBr (Fig. 7s) confii the location of the methionines. Pool F of this experiment contained a pure polypeptide. The NH&erminal residue was lysine and the amino acid composition corresponded closely to residues 2 to 26 except that the methionine had been converted to homoserine by CNBr (data not shown). Pool A of Fig. 7s contained the polypeptide arising from residues 27 to 88 and was further purified by chromatography on Sephadex G-75 (superfine) using 9% formic acid. A limited sequenator run was done which gave the sequence Gly-Thr-Ile-Leu-Phe-Gly corresponding to that of residues 27 to 32 and the amino acid composition corresponded quite closely to that of residues 27 to 88, but with one homoserine present (data not shown). The fourth polypeptide, CB-8B (residues 91 to 123), will be considered below. Thus, these results provided confirmation of the 4 The abbreviations used are: HPLC, high pressure liquid chromatography; BCT, biocytin; gas-liquid chromatography; chloromethyl ketone.

DMBA, N,N’-dimethylbenzylamine; TPCK, L-1-tosylamido-2-phenylethyl-

GLC,


I

45

75

80 - Gln - Thr

- Lys - Ala

30

70

- Gly

Val

15

Sequence Amino Residue

1-123 1.3SE Obs I Calc

Met

T-1B

I

3.6

4

4

1.1

10

10

1 4.0

123

123

finding

T-4A

of

Obs Calc

11617

I subunit

and ofpolypeptides

78-101 T-6F BY sequ

Obs

Calc

BY sequ

1

2

1.2

1

2

1.0

1

2

4

4

2.8

3

3

1.x

2

2 4

80

79

47

50

1

arginine

in

this

fraction

Polypeptide

IUIlOl

Biotin subunit

613

1-123

1-55

DMBA

150

1-123

l-47

T-4A T-1B T-6F CB-8B TS-SC

300 650 100 150 125

43-121 52-101 78-101 91-123 111-123

Sequenced

43-82 52-89 78-97 91-115 111-123

22

remains

Conditions "F;ge;p

Subunit

PrOgram (protein)

Yes

No

0.1 M Quadrol

Yes

Yes”

Ye2

0.1 M Quadrol DMBA (peptide) DMBA (peptide) DMBA (peptide) DMBA (peptide)

Yes No Yes Yes Yes

Yes’ Yes Yes’ Ye8 Yes

Yes” No Yes/ Yesh Yes’

39,

residue

65,

presence of methionine at residues 1, 26, 88, and 90. In our preliminary reports, residue 35 was not given although it was tentatively identified at that time as threonine. This residue was definitely identified as threonine by HPLC and GLC in the more recent degradation of the biotinyl subunit. The alanine at residue 38 was identified in one degradation but not in the other. This residue is the only one in the sequence which was identified by only one procedure. The sequence from residue 43 to 89 was established in Fragments T-4A and T-1B but residue 81 was not identified in either of these degradations. For this purpose, the polypeptide representing residues 78 to 101 (T-6F) was generated from Fragment T-4A by tryptic digestion (Fig. 6s). Analysis of this polypeptide identified threonine at residue 81 by both

CB-8B

TS-9C

3.4 5.1

3 6

3 6

33

33

Obs

Calc

BY sequ

1.1 2.1

1 2

1 2

13

13

unexplained.

TABLE II of sequenator analysis PolyGLC HPLC brene

residue

111-123

BY Obs I Calc I sequ

24

No

n Through residue 27. h Both Waters and Durrum systems were used through thereafter Waters only. ’ GLC was discontinued after residue 77. d Both Waters and Durrum systems were used through thereafter Waters only. ’ GLC was discontinued after residue 92.

91-123

Tentative

identification

Asn 24, Thr 28, Thr 35, Gly 48 Gly 36, Pro 39

Va176 Ala 78, Thr

Not identified Lys 51, Glu 54 Met 26, Ala 38, Arg 42, Ala 44, Gly46 Gly 79, Thr 81 Thr 81, Glu 86

92, Ile 94

I Only Waters system was used with HPLC. p GLC was discontinued after residue 114. h Both Waters and Durrum systems were used through residue 112, thereafter Waters only. ’ HPLC with both Waters and Durrum systems were used throughout.

GLC and HPLC. Also, with this polypeptide, the sequence surrounding the biocytin was fnmly established. In our preliminary studies we misinterpreted results from the fraction obtained by CNBr cleavage and reported the sequence Bet-Thr-Glu-Ile--whereas the sequence (from analysis of T-6F) is clearly Met-Bet-Met-Glu-Thr-Glu-Ile---. Apparently, the CNBr cleavage was incomplete, yielding BctHser-Glu-Thr-Ile--as a minor contaminant of the polypeptide Glu-Thr-Glu-Ile---. It was known at that time that a methionine preceded the biocytin and we expected by CNBr cleavage to obtain a polypeptide containing an NHz-terminal [3H]biocytin. The first sequencing cycle of the impure fraction yielded the expected radioactivity and some glutamic acid. This radioactivity from the minor contaminant was taken to


The

52-101

BY Calc I sequ

TABLE of biotinyl

composition

43-121

Obs

9.8

Total

*

BY sequ

acid

of Biotinyl

11618


5 This portion of the sequence of biotinyl subunits has been a source of diffkulty for others. We were provided with preliminary information concerning the sequence of the biotinyl polypeptide obtained from acetyl-CoA carboxylase of Escherichia coli but only one of the two methionines following the biocytin had been detected at that time (Fig. 2 of Ref. 12). “Leu-Val-Lys-Glu at residues 106 to 109 (previously 102 to 105) was reported incorrectly as Glu-Lys-Val-Leu as a result of typographical errors.

enzymes during the isolation of the transcarboxylase biotinyl subunit from the transcarboxylase.

or of the

DISCUSSION

The amino acid composition of the biotinyl subunit of transcarboxylase is unusual since there are no cysteine or tryptophan residues, only 1 each of histidine, phenylalanine, and tyrosine, and 49 of the 123 residues are glycine, alanine, or valine. There are no other complete sequences of biotinyl subunits from biotin enzymes to compare with that from transcarboxylase. However, a substantial portion of the biotinyl subunit from acetyl-CoA carboxylase of E. coli has been determined (12). In Fig. 3, the two sequences are compared by aligning the biocytins of the polypeptides. The two enzyme subunits bear weak evidence of homology, including the identical pentapeptide containing the biocytin. A major difference is the cysteine-leucine exchange at residue 83. A study, by circular dichroism, of the biotinyl subunit of acetyl-CoA carboxylase and derived polypeptides has shown that cysteine-83 is very important in the structure of the biotinyl subunit of acetylCoA carboxylase (13). Rylatt et al. (14) have determined the sequence of three biotinyl polypeptides from pyruvate carboxylases of the livers of sheep, chicken, and turkey. The two avian polypeptides were found to be identical and the avian and sheep polypeptides highly homologous. The sequences of the biotinyl polypeptides of the two pyruvate carboxylases are compared in Fig. 4 with those of acetyl-CoA carboxylase of E. coli and of transcarboxylase. The sequence from transcarboxylase is more similar to that of pyruvate carboxylase than to that of acetylCoA carboxylase, perhaps because transcarboxylase and pyruvate carboxylase have in common Partial Reaction lb. However, acetyl-CoA carboxylase and pyruvate carboxylase have Partial Reaction la in common and there is considerably less homology between these two sequences. Perhaps, the portion of the biotinyl subunit in the immediate vicinity of the biocytin is quite directly coordinated with the keto acid site. It is possible that a portion more remote from the biocytin of the biotinyl subunit is more directly coordinated with the ATP-CO2 site and that, in this portion, there is homology between the biotinyl subunit of acetyl-CoA carboxylase and the sequence of pyruvate carboxylase. There is an Ala-Met-Bet-Met sequence in all four polypeptides. This sequence may form part of the recognition site for holoenzyme synthetase which biotinates the apoenzyme. McAllister and Coon (15) found that the holoenzyme synthetases from rabbit liver, yeast, and P. shermanii add biotin to apopropionyl carboxylase from rat liver, apomethylcrotonylCoA carboxylase from Achromobacter, and apotranscarboxylase from P. shermanii. Perhaps the identical tetrapeptide portions of the sequences have been conserved in biotin enzymes to provide the microenvironment required for recognition by holoenzyme synthetase of the specific lysine to be biotinated. The ultimate goal of our studies is to relate the structure of the biotinyl subunit to its functions. It is known that residues 1 to 42 of the biotinyl subunit are sufficient to link the outside 5 SE subunit to the central 12 Sn subunit (7). Variable distances have been observed between the outside and central subunit by electron microscopy (16, 17) and it has been proposed that one portion of residues 1 to 42 combines with the outside subunit and another with the central subunit and a portion between permits flexibility and variability of the distance between the linked subunits. A notable feature of this portion of the sequence is the alternating aspartic acid and valine residues from residues 13 through 19. Calculation


indicate that the first residue was biocytin, while the glutamic acid was considered to arise from a contamination. The second cycle gave threonine which was considered to arise from the residue following the biocytin. Thus, the sequence of the major peptide was observed but Bet was reported instead of Glu and the methionine and glutamic acid following the biocytin were overlooked.” The location of the biocytin and methionine-90 has been confinned by manual degradation (11) of a polypeptide (T(u) consisting of residues 89 to 101 (data not shown). The bond between methionine-88 and biocytin-89 was apparently cleaved by trypsin and Fragment T-c* was obtained rather than T-6F. This anomalous cleavage was later avoided by pretreatment of the trypsin with TPCK and dithiothreitol as in the experiment of Fig. 5s. A similar cleavage of methionylbiocytin was observed by Sutton et al. (12) in generating the biotinyl polypeptide from acetyl-CoA carboxylase of Escherichia coli. Manual degradation of peptide T-LY released the radioactivity in the fist cycle and thereafter Met-Glu-ThrGlu-Ile. The COOH-terminal residue was identified as GlyLys. With carboxypeptidase B at pH 8, only lysine was formed in 1 h and glycine was released after 2 h. Both were identified as the dansyl derivatives. Thus, these results were in complete agreement with the sequence shown in Fig. 1. The COOH-terminal portion of the sequence was determined using polypeptides CB-8B (residues 91 to 123) and TS9C (residues 111 to 123). As explained above, in our preliminary studies (6, 8-10) we had examined a mixture of peptides obtained by CNBr cleavage. The sequence reported from threonine-92 to valine-109” was the same as that found by degradation of CB-8B except that serine was reported instead of aspartic acid at residue 99. The aspartic acid from CB-8B has now been identified by HPLC using both the Waters and Durrum systems and by GLC. The remainder of the sequence was reported as Gln-Lys-?-Ala, which corresponds to residues 110 to 113, and was in error. When it was established by degradation of polypeptide CB8B that arginine is residue 110, it became obvious that the COOH-terminal portion of the sequence 111 to 123 could be obtained by trypsin treatment of the succinylated biotinyl subunit (Fig. 9s). The degradation of this polypeptide (TS9C) proceeded exceptionally well, giving the entire sequence, and each residue was identified by HPLC using both the Waters and Durrum systems and by GLC. This sequence overlapped that determined with polypeptide CB-8B and, thus, established its location and confirmed the placement of arginine at residue 110. In addition, the Lys-Ile-Gly sequence at the COOH-terminal end was established by carboxypeptidase degradation using carboxypeptidase Y and B in succession. With carboxypeptidase Y (pH 5.5), in 2 h glycine was identified as the COOH-terminal residue and isoleucine as the penultimate residue. By further incubation with carboxypeptidase B at pH 8.0 lysine was released. The major uncertainty concerning the sequence is the nature of the short form of the biotinyl subunit (6). Preliminary results with carboxypeptidases suggest that the COOH-terminal residue of the short form is leucine (residue 119). The short form probably arises due to the action of proteolytic

Subunit

Sequence

of Biotinyl

41

50 Ala-Gly-Gly-Ala-Gly-Ala-Gly-Lys-Ala-Gly-Glu-Gly-Glu-Ile-Pro-Ala-Pro

Transcarboxylase P. shermanii -~ Acetyl-CoA --E. coli

Subunit

11619

60

carboxylase

61 Leu-Ala-Gly-Thr-Val-Ser

70 Ile-Leu-Val-Lys-Glu

Thr-Pro-Se--Pro-Asp-Ala

Ala-Phe-Ile-Glu-Val

91

Glu-Thr-Glu Met-Asn-Gln

121 ly-COOH lu-COOH

FIG. 3. Comparison of the CoA carboxylase of molecular aligned at the biocytin.

Pyruvate sheep

carboxylase:

Pyruvate avian

carboxylase:

Transcarboxylase: P. shermanii Acetyl-CoA carboxylase: g. coli

FIG. 4. Comparison carboxylase (12). The

of the transcarboxylase 9100 (12). The numbering

biotinyl system

subunit with a portion is from the transcarboxylase

of the biotinyl subunit of acetylsubunit and the polypeptides are

1

*

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Gl-/

Gin

Pro

Leu

Val

Leu

Ser

Ala

Met

Bet

Met

Glu

Thr

Val

Val

Thr

Gly i-l-

Ala

Pro

LeU

Val

Leu

Ser

Ala

Met

Bet

Met

Glu

Thr

Val

Val

Thr

-;Thr(

Val

Ala

Met

Bet

Met

Glu

Thr

Glu

- -1 ;Ile,

Asn

Gin

Asn

I ' I 1 (Thr, Leu --

I Cys

Ile

I ,

Val

of the sequence adjacent to biocytin polypeptides are aligned at the biocytin.

for

by the Chou-Fasman method (18) indicates the potential for p sheets between residues 3 and 6 and between 28 and 31. Wood and Zwolinski (6) have suggested that the P-sheet regions may provide the separate binding sites for the outside and central subunits. They also propose that there may be ionic interactions via the aspartic residues which are broken during the negative staining for electron microscopy and that this is responsible for the observed variation of the distance between the outside and central subunits. In addition to providing the binding sites for the outside and central subunits, the biotinyl subunit has a role in the orientation of the biotin so that it can serve as a carrier of the carboxyl groups between the keto acid sites on the outside subunits and the CoA ester sites on the central subunit. It seems likely that the biotinyl subunit bends back on itself for the placement of biotin between these two subunits. This suggestion follows from the fact that a portion of the sequence at residues 42 and 43 apparently is exposed, since this bond is readily cleaved by trypsin, leaving the outside and central subunits still attached. However, a portion of the sequence containing the biotin must be between the outside and central subunits so as to serve as a carboxyl carrier between the keto acid and CoA ester sites on these two subunits. Fung et al. (19) by NMR and EPR techniques have estimated the distance between the CoA and keto acid sites of transcarboxylase using a nitroxide analogue of propionyl-CoA and calculated that the bound propionyl-CoA analogue is 7.9 A from the

Met

pyruvate

Bet

Met

Met

carboxylases

Asn

(14),

Gln

I

I

1

I

IIle Clu '- - 1

17

18

19

Al-al

Asn

Lys

transcarboxylase,

and

acetyl-CoA

methyl protons of the bound pyruvate. It has been proposed (6) that the Pro-Ala-Pro sequences at 39 to 41 and at 58 to 60 may provide the turns which permit the biotinyl subunit to bend on itself and insert the biotin between the two subunits. This proposal is based on the well known fact that prolines produce bends in polypeptides. The sequence requirement for the proper orientation of the biotin apparently is quite rigid. C. Bahler and H. G. Wood7 have shown that polypeptides T-1B and T-6F are effective in restoring partial catalytic activity of 26 S transcarboxylase which has been made inactive by treatment with trypsin to remove polypeptide T-4A. However, neither the 19-residue biotinyl polypeptide from avian pyruvate carboxylase (Fig. 4) nor the biotinyl polypeptide from acetyl-CoA carboxylase (Fig. 3) is effective in restoring this activity. (We wish to thank D. B. Keech, University of Adelaide and R. R. Fall, University of Colorado, for gifts of these polypeptides.) By the application of the new computerized Chou-Fasman method (18), two regions, 66 to 72, and 80 to 85, are predicted to undergo LY,,5 transition. By changing from one conformation to the other, the two regions may serve as the “hinge” for movement of the biocytin between the central and outside subunits. If, in these calculations, one uses lysine for the biocytin, then the sequence around the biocytin (residues 80 to 93) is predicted to be potentially a strong (Y helical region. 7 C. Bahler

and H. G. Wood,

unpublished

results.


Enzyme

sequence weight


11620

This also holds true for the biotinyl polypeptides of pyruvate carboxylase (14) and of acetyl-CoA carboxylase. There is predicted to be a p turn in residues 97 to 100 and another region of (Y helix starting at residue 103 and extending to residue 112 followed by another ,L?turn at residues 115 to 118. Similar calculations have been made by Rylatt et al. (14) for the biotinyl polypeptide of pyruvate carboxylase. It will be of interest to determine whether these predictions from the Chou-Fasman (18) criteria are confirmed by measurement of circular dichroism and by x-ray crystallography. Achnowledgments-We wish to thank Professor Hans Neurath for helpful discussions and support and Professor Robert Becker for the assistance and facilities provided to H. G. W. during a portion of these studies. REFERENCES

14. 15. 16.

P. R., and Bradshaw, R. A. (1977) J. Biol. Chem. 252, 39343940 Fall, R. R., Glaser, M., and Vagelos, P. R. (1976) J. Biol. Chem. 251,2063-2069 Rylatt, D. B., Keech, D. B., and Wallace, J. C. (1977) Arch. Biochem. Biophys. 183, 113-122 McAllister. H. C.. and Coon. M. J. (1966) J. Biol. Chem. 241, 2855-2861 Green, N. M., Valentine, R. C., Wrigley, N. G., Ahmad, F., Jacobson. B.. and Wood. H. G. (1972) J. Biol. Chem. 247.62846298

17. Wrigley, N. G., Chiao, J.-P., and Wood, H. G. (1977) J. Biol. Chem. 252,1500-1504 18. Chou, P. Y., and Fasman, G. D. (1978) Aduan. Enzymol. 47, 45148 19. Fung, C. H., Gupta, R. K., and Mildvan, A. S. (1976) Biochemistry 15,85-92 20. Dickerson, R. E., and Geis, I. (1969) in The Structure and Action of Proteins (Dickerson, R. E., and Geis, I., eds), Chap. 1.4, Harper and Row, New York 21. Zwolinski, G. K., Bowien, B. U., Harmon, F., and Wood, H. G. (1977) Biochemistry 16, 4627-4637 22. Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H., and Walsh. K. A. (1972) Biochemistrv 11. 4493-4502 23. Brauer, A. W., Margohes, M. N., and Haber, E. (1975) Biochemistry 14, 3029-3035 24. Hermodson, M., Schmer, G., and Kurachi, K. (1977) J. Biol. Chem. 252, 6276-6279 25. Tarr, G. E., Bucher, J. F., Bell, M., and McKean, D. J. (1978) Anal. Biochem. 84, 622-627 26. Bridgen, P. J., Cross, G. A. M., and Bridgen, J. (1976) Nature (London) 263, 613-614 27. Ericsson, L. H., Wade, R. D., Gagnon, J., McDonald, R. M., Granberg, R., and Walsh, K. A. (1977) in Solid Phase Methods in Protein Sequence Analysis, Institut National de la Sante et de la Recherche Medicale Symposium No. 5 (Previero, A., and Coletti-Previero, M. A., eds), P. 137. Elsevier/North Holland Press, Amsterdam 28. Wood, H. G., Lochmuller, H., Reipertinger, C., and Lynen, F. (1963) Biochem. 2. 237, 247-266


1. Chuang, M., Ahmad, F., Jacobson, B., and Wood, H. G. (1975) Biochemistry 14, 1611-1619 2. Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974) J. Biol. Chem. 249, 6657-6667 3. Alberts, A. W., and Vagelos, P. R. (1972) in The Enzymes (Boyer, P. D., ed), 3rd Ed, Vol. 6, p. 37, Academic Press, New York 4. Wood, H. G., Chiao, J.-P., and Poto, E. M. (1977) J. Biol. Chem. 252, 1490-1499 5. Wood, H. G., Ahmad, F., Jacobson, B., Chuang, M., and Brattin, W. (1975) J. Biol. Chem. 250,918-926 6. Wood, H. G., and Zwolinski, G. K. (1976) Crit. Reu. Biochem. 4, 47-122 7. Ahmad, F., Jacobson, B., Chuang, M., Brattin, W., and Wood, H. G. (1975) Biochemistry 14, 1606-1611 8. Zwolinski, G. K., Bowien, B., Wood, H. G., and Ericsson, L. (1975) Fed. Proc. 34, 631 9. Wood, H. G. (1976) Fed. Proc. 35, 1891-1907 10. Wood, H. G., and Barden, R. E. (1977) Anna. Rev. Biochem. 46, 385-413 11. Bruton, C. J., and Hartley, B. S. (1970) J. Mol. Biol. 52, 165-178 12. Sutton, M. R., Fall, R. R., Nervi, A. M., Alberts, A. W., Vagelos,

13.

Subunit

Sequence

w.

Lee

Haby,

Both0

“.

mwien,

Gene

K.

Z”olinski,

K.

of Biotinyl

11621

Subunit

Ganesh

Materials form of transc.+rboxyiase containing [%]blotin as previously described (4). Other materials were WC@-trypin from Worthington Biochemical Corp.; dii60pTOpylflUOrOphoSphate treated carboxypeptidase B from Emehringer Mwmheim; carboxypeptidase Y from Calbiochem., Inc.; dansyl chloride, standard dansyl amino acids, phenylisothiocyanate, trifluoroacetic acid and "Sequanal" grade pyridine from Pierce Chemical Company; phenylmethyl sulfonyl fluorde, diisopropylfluorophosphate. dithiothreitol and guanidine.HCl from Sigma Chemical CO.; suxxn~c anhydride and cyanogen bromide from Eastman Kodak Co.; TFCK from Calbiochemi fluorescamine from Rock Diagnostics, urea from Mallinckrodt Chemical Co.; Ultrex grade EC1 fr0m'J.T. Baker Chemical Co.; Sephadex gel filtration media from Pharmacxa; DEAE-cellulose ion exchange resins from Whatman; polyamide thin layer plates from Schleicher and Schuell and the deionizing filter for urea from Crystalab. All other solvents and chemicals were reagent grade or better.

The was prepared as follows:

265

pOlyacrylamide bands contain acid analysis the C-terminal are called

Purification plypeptides Pool

by from B

Trypsinization 26s transcarboxvlase

tion

of

sodium methyl with

the

acetate, sulfcmyl same

preparative the trypsinization polypeptide

contained of

265

transcarboxylase was done

pH 5.5, containing fluoride and 0.2% buffer (Fig. ZS).

gel

electrophoresis of I*S

of tran.xar-

T-10.

at

10m4k sodium

oH

5.5

EDTA. azide,

The in

10e4 and

trypsinizaorder

to

M phenylwas eluted

gel electrobiotin and shows there end of the the long and

Trypsinization of 18s transcarboxylase and isolation of the resulting biotinyl polypeptides - Ahmad et al. (7) found that brief trypsinization of 18s transcarboxylase at pH 6.3 in phosphate buffer yields a 66 residue biotinyl polypeptide and smaller polypeptides while the major oortion of the transcarboxvlase remains intact beina held tcmether bv another DOTtion of the biotinyl subunit. This proc&ure ha; been uset to obtain biotinyl wlypeptides for the sequence studies. The 18s transcarboxylase was trypsinized and the biotinyl plypeptides were separated from the resulting protein by chromatography on Sephadex G-50 (7). The hiotinyl plypeptides were then purified (Fig. 1s) by preparative gel electrophoresis (Canalco chamber from Central Industrial Corp., Rockville, Md.) as described by Zwolinski et al. (22). The plypeptide in Pool B which was reported to contain 47 residues (6) actually con tains 50 residues. This plypeptide is designated T-1B in this publication.

FRACTION a tryptic ml.

Fig.

zs. digest

NUMBER

Fractionation of the biotinyl polypeptide from of 265 transcarboxylase. Fractions were 1.8


major bands of varying intensity on phoresis (51. The proteins of both have a methionine N-terminal. Amino is a small difference presumably at plypeptide chain. They, therefore, Short forms of the hiotinyl subunit.

Fig. 1s. biotinyl boxy1ase.


11622

Subunit

40 FRACTION

t?MBER

FRACTION

NUMBER


FF%TlON

80 NUMBER