Eur. J. Biochem. 72,425-442 (1977)

The Primary Structure of Yeast Alcohol Dehydrogenase Hans JORNVALL Department of Chemistry, Karolinska Institutet, Stockholm (Received April 14/September 9, 1976)

Eight different types of peptide mixtures from [14C]carboxymethylated yeast alcohol dehydrogenase were obtained using trypsin with or without prior maleylation of the substrate, chymotrypsin, pepsin, microbial proteases or CNBr. Each mixture was fractionated by exclusion chromatography and peptides were further purified on paper. From results of analyses of all fragments it seems possible to deduce a primary structure of 347 unique residues in three segments. Together, the segments can account for the whole protein monomer with the exception of a small connecting region. Many unfavourable structures complicated the determination and made single sequence conclusions tentative, but known data are consistent and for most segments of the monomer results are abundant. Several microheterogeneities in the protein are indicated and one apparent amino acid exchange is characterized, suggesting that different types of subunits occur. This may probably be correlated with genetic polymorphism in yeast. Multiple desamidations are also characterized and a few of these affect particularly labile structures. Many residues are unevenly distributed and unexpected patterns are shown. Elements of repetitive sequences occur, reducing the uniqueness of structures. Hydrophobic segments are found, and the uncharacterized region is, at least in some subunits, in a core-like tryptic segment. These and other aspects of the structure may explain some properties of the monomer, and form the background for evolutionary, structural and functional correlations with releated enzymes. ing structural and functional residues may be traced, suggesting similarities in tertiary structures and enzymic mechanisms [6]. The primary structure of the yeast enzyme is of interest for correlation with the known primary [7,8] and tertiary [9] structures of mammalian alcohol dehydrogenases in order to explain the observed differences and reveal relationships between structure and function. The primary structure is also necessary for tracing the enzyme's evolution, which apparently is rapid compared to that of some other dehydrogenases [2,8]. From this aspect yeast alcohol dehydrogenase is of further interest, since in quaternary structure, subunit size and to some extent zinc content it resembles other types of dehydrogenases [2] rather than mammalian alcohol dehydrogenases, and thereAbbreviations. Dansyl, 5-dimethylaminonaphthalene-1-sulpho- fore may conceivably form a link between proteins, nyl; Tos-PheCHzC1, L-1-tosylamido-2-phenylethylchloromethyl further illustrating the similarities among dehydroketone; Tos-LysCHzCl, l-chloro-3-tosy~amido-7-amino-2-heptagenases observed in their tertiary structures [lo- 121. none. In the present work, many different types of pepEnzymes. Yeast alcohol dehydrogenase (EC 1.1.1.1); horse liver alcohol dehydrogenase (EC 1.1.1.1); glyceraldehyde-3-phostides from the ['4C]carboxymethylated protein were phate dehydrogenase (EC 1.2.1.12); chymotrypsin (EC 3.4.21.1); studied in order to determine the amino acid sequence trypsin (EC 3.4.21.4); kallikrein (EC 3.4.21.8); elastase (EC 3.4.21. of the monomer. Results are consistent with previous 11); pepsin A (EC 3.4.23.1); thermolysin (EC 3.4.24.4); Staphyloreports containing incomplete information on part of coccus aureus extracellular protease I (EC 3.4.99. -); Myxohacter the structure [2,13 - 191, and permit important correlaAL-1 extracellular protease I1 (EC 3.4.99. -).

Yeast alcohol dehydrogenase is a tetramer with a molecular weight of about 150000 [l]. Its subunit is distantly related to that of the dimeric mammalian alcohol dehydrogenases but these two types of proteins are also highly different. Thus, a structural comparison after analysis of part of the yeast enzyme has indicated a maximal identity of only 40% [2]. The enzymes differ in subunit size and quaternary structure (cf. [2]), and the active yeast enzyme has been reported to contain one [3] or two [4] zinc atoms per subunit against always two in the native horse liver enzyme [I]. Substrate specificities, turnover numbers and other enzymic properties are also different [ 1,5]. Nevertheless, mammalian and yeast alcohol dehydrogenases are probably evolutionarily linked [2], and correspond-

426

tions with properties of the enzyme and with structures of related proteins. The support for the amino acid sequence deduced and for postulated amino acid heterogeneities and chemical modifications is given in this report, together with such structural characteristics and interpretations which are immediately available. Further structural, functional and evolutionary relationships, which are revealed by different comparisons involving the present structure, are given in the accompanying analysis [20]. MATERIALS AND METHODS Protein Material and Pretreatments

Yeast alcohol dehydrogenase was obtained from Boehringer Mannheim GmbH, as a crystalline suspension in 2.4 M ammonium sulphate, and had been prepared from Saccharomyces cerevisiae (supplied by BAST GmbH, Niirnberg, Germany) following an extension of a previous procedure [21]. The protein was radioactively labelled by [14C]carboxymethylationand therefore first thoroughly dialyzed against distilled water, initially containing a few drops of ammonia, to remove salt. The lyophilized protein was then dissolved (10 mg/ml) in 6 M guanidine-HC1, 0.1 M Tris, 2 mM EDTA, pH 8.1, reduced with dithiothreitol (about 50 pg/mg protein, i.e. 50% molar excess of dithiol reagent over protein thiol groups) for 2 h at 37 "C, and carboxymethylated with neutralized iodo[2-'4C]acetic acid (170 pg/mg protein, i. e. 15 % excess over total - SH groups) for 2 h at 37 "C. All reactions were performed in a nitrogen atmosphere and reagents were subsequently removed by dialysis against distilled water. Samples for treatment with CNBr were carboxymethylated in a milder way for 1 h at 25 "C using less dithiol and iodoacetate (about 75%) in order to avoid extensive alkylation of methionine [22]. Specific radioactivity was about 1-2 Ci/mol, which, in the purification steps on paper, produced suitable autoradiographs in just a few days of most radioactive peptides, including those that were recovered in low yield. Maleylations [23] and citraconylations [24] were performed at room temperature in 8 M urea, 0.1 M sodium pyrophosphate with successive additions of NaOH and anhydride to give a 50-fold final excess of reagent over total protein amino groups. Subsequently, 0-maleyl groups [7] were removed by addition of hydroxylamine-HC1 (and NaOH ; constant pH) to give a final concentration of 0.4- 0.8 M [25]. Enzymes and Proteolytic Cleavages

Tos-PheCHZCl-trypsin, chymotrypsin and pepsin were obtained from Worthington Biochemical Corporation. Elastase was from Sigma Chemical Co and

Structure of Yeast Alcohol Dehydrogenase

thermolysin from Chugai Boyeki Co (Osaka, Japan). The chymotrypsin preparation was treated with TosLysCHzCl before use [26]. A staphylococcal protease with high specificity for glutamyl bonds [27 - 291 was prepared and kindly supplied by Prof. Philipson and coworkers (Dept of Microbiology, Uppsala University), and by Dr Bjorklind (Dept of Bacteriology, Karolinska Institutet). A Myxobacter protease, preferentially cleaving at the N-terminal side of lysine [30], was a kind gift from Prof. Wolfe (Dept of Microbiology, University of Illinois), and pancreatic kallikrein was a gift from Prof. Werle (Institute for Clinical Chemistry and Biochemistry, University of Munich). The protein substrate was digested at concentrations of about 3 mg/ml with enzyme (1 : 100, by weight) at 37 "C for 4 h in 0.1 M ammonium bicarbonate for trypsin, chymotrypsin, elastase, kallikrein and the staphylococcal protease, in 5 % formic acid for pepsin, in 0.02 M Tris, pH 9, for the Myxobacter protease, and in 0.2 M sodium acetate, 5 mM CaC12, pH 8.5 for thermolysin. In the case of partly insoluble digests, treatments were repeated as indicated. Secondary digestions of small peptides were performed with 10-100 pg of enzyme in 0.1-1 ml of the buffers. Cleavage with CNBr at a 10-15-fold weight excess was carried out in 70 % formic acid at room temperature. After all proteolytic treatments, peptide mixtures were fractionated immediately or frozen and lyophilized . Pep tide Purifications

Peptide mixtures for preparative digests were first fractionated by exclusion chromatography on Sephadex G-50 fine (2.5 x 100 cm or 5 x 100 cm) in 0.1 M ammonium bicarbonate for enzymic digests and in 25 - 50 % acetic acid for CNBr fragments. In the case of partly insoluble material, pyridine or ammonia (enzymic digests) or urea to 8 M (CNBr peptides) was added before application. Alternatively, CNBr peptides were citraconylated [31]. Eluted material was measured for absorbance at 254 nm in an LKBUvicord flow cell or at 280 nm in a Zeiss PMQ I1 spectrophotometer, and for radioactivity in a NuclearChicago scintillation flow cell or a Frieseke & Hoepfner gas-flow counter. Pooled fractions were lyophilized, and were usually purified further on paper by high-voltage electrophoresis at pH 6.5, 3.5 and 1.9, and by chromatography in butan-1-ol/acetic acid/water/pyridine, 15/31 12/10, by vol., as previously described [22]. Material was localized by staining appropriate guide strips with different reagents [22], by autoradiography, or by staining the whole papers with fluorescamine (Fluram-Roche) in acetone (4 pglml). Peptides were eluted with distilled water.

H. Jornvall

Analytical Methods

Total compositions were determined on Beckman 120B or 121M amino acid analyzers. Peptides were hydrolyzed for 20- 24 h at 110 "C with 6 M HCl containing 0.1 % mercaptoethanol. Alternatively, for small peptides only, hydrolysates were esterified with butan-l-o1/3 M HC1, acylated with trifluoroacetic anhydride and analyzed by gas chromatography [32] on a Hewlett-Packard 402 gas chromatograph. For analysis of the whole protein, hydrolysis was for 20 h, 48 h and 72 h using triplicate samples for each time. Serine and threonine values were extrapolated to zero time, valine and isoleucine values were calculated from the 72-h values, and contents of remaining residues from the averages. Sequence analysis was largely performed with the dansyl-Edman method and dansyl amino acids were identified by thin-layer chromatography as previously described [33]. A short region of a large peptide was also analyzed in a Beckman liquid-phase sequencer using quadrol (N,N,N,N-tetrakis(2-hydroxypropyl)ethylene diamine) in a modified protein program. '4C-labelled residues removed during Edman degradations were always checked by measurements of radioactivity in the extracts. Amide groups were determined from the electrophoretic mobilities of peptides [34]. Nomenclature of Peptides

Peptides are identified by a combination of letters and figures. T, C, P, R, M, B, H, Mal, or HC1 indicate that the fragment was obtained by treatment with, respectively, trypsin, chymotrypsin, pepsin, the staphylococcal protease, the Myxobacter protease, CNBr, thermolysin, maleylation plus trypsin, or limited acid hydrolysis. In the case of multiple treatments, the first letter tells the initial method used and the second letter the method for redigestion. Numbers are consecutive and denote the order in the protein chain of those peptides recovered in each type of digest (first number), or in the case of redigestions, the order of peptides within a larger fragment (second number). RESULTS PEPTIDE SEPARATIONS

Eight different types of peptide mixtures from ['4C]carboxymethylated yeast alcohol dehydrogenase were analyzed on a preparative scale using 100250 mg protein each time. They were obtained by treatment with trypsin, chymotrypsin, pepsin, a staphylococcal protease, a Myxobacter protease, a combination of these proteases, CNBr and trypsin subsequent to maleylation, respectively. Most digests were performed and analyzed more than once. Diges-

427 Table 1. Summary of'results from preparative digests studied in the sequence work on yeast alcohol dehydrogenase ~

~~

Proteolytic treatment

Number of pure peptides, including fragments from redigestions

Proportions of protein recovered in pure peptides

Proportions of protein sequenced in pure peptides

Trypsin

92

88

Chymotrypsin

64

84

71

Pepsin

44

61

35

Staphylococcal protease 34 soluble peptides 18 precipitate redigested with Myxobacrer protease 16

51 31

40 21

36

22

_ _ _ ~ __ 16

Myxohacter protease

12

23

8

Trypsin on rnaleyated substrate

21

79

26

CNBr

17

31

16

tions with elastase and kallikrein were also attempted but found to be of no value (elastase, only oligopeptides; kallikrein, no proven cleavage). In addition to the enzymes used for preparative digests, thermolysin or limited acid hydrolysis was valuable for redigestions of isolated peptides, as indicated. The results of the different digests are summarized in Table 1. Each preparative digest was first fractionated by exclusion chromatography on Sephadex G-50. Elution patterns are shown in Fig. 1. The peptide mixtures obtained with trypsin, the Myxobacter protease or CNBr were not completely soluble but could be submitted to gel chromatography in 0.1 M ammonium bicarbonate after solubilization with small amounts of concentrated ammonia, added just before application to the column. Alternatively, in some preparations of CNBr peptides, these were first citraconylated [31], or instead separated by fractionation in 25 - 50 % acetic acid with essentially similar elution profiles (cf. Fig. 1H). The digest obtained with the staphylococcal protease was highly insoluble even after repeated additions of enzyme, and was therefore divided by centrifugation, repeated washings and recentrifugations. The pooled supernatants were then fractionated separately (Fig. l D) whereas the precipitate was redigested with the Myxobacter enzyme before fractionation (Fig. 1 E). Peptides in all fractions were purified further by repeated steps of paper elctrophoresis and chromatography as given in the Methods section. The fraction from the exclusion chromatographies (Fig. 1) used for

Structure of Yeast Alcohol Dehydrogenase

428

,

Effluent (ml)

iqo

2yo

3T

5:

4vO

1

Effluent (ml)

900

700 1 . 2 p

*

I

I

1100

1300

I

1500

1700

1

r

a

8

1

8

E

Fraction number

Fraction number

Effluent (mi) Effluent (mi)

200

100 1 .6

-

1 .2

-

1

2 000

500

400

I

-

1 .4

1500

loo0

500

300

.o -

0.8 0.6 -

0.4

-

0.2

-

0 d+

6

5

1 2 3 4

" I

7

1

3

2

Fraction numbei

4

Effluent (ml)

800

-

1000

1200

1400

1600

7

3 4 5 6

2

8

9

500

1800

1

1

5

6

7

8

Fraction number

o r

'

900

700 '

'

'

'

Effluent (mi) 1100 1300 '

'

'

'

1700

1500 '

'

'

'4

'

10

Fraction number

1

Effluent (ml)

r""""""""' 400

-

"

I,

2

3

1

2

3

4

5

6

Fraction number

1

5

6

7

8

9

10

11

Effluent ( m i )

i"

7

4

Fraction number

600 800 loo0 1200 1400 1600 1800 2000

700 900 "

"

1100 1300 1500 1700 1900 "

"

"

8 Fraction number

"

"

I

H. Jornvall

purification is given in the sequence tables by a number in parentheses following the name of the peptide. The nomenclature of peptides indicates the relevant chromatogram for the fraction number as well as the proteolytic treatment used. Electrophoretic mobilities at pH 6.5 of important fragments are listed in the data tables.

THE STRUCTURE OF AN N-TERMINAL 172-RESIDUE SEGMENT

All peptides purified from this region as well as the results of sequence analyses are shown in Table 2. Compositions, recoveries and electrophoretic mobilities of all important peptides are given in Table 3. Three large fragments were purified from the Nterminal half of the protein. Their total compositions as determined by acid hydrolysis are shown in Table 4 and are in excellent agreement with the compositions obtained from the sum of sequence results of constituent small peptides. This is found for the 69residue tryptic peptide T13, the 70-residue CNBr fragment containing residues 99 - 168 and the entire 172-residue maleylated tryptic peptide. The CNBr fragment is obtained from repeated exclusion chromatographies of fraction 1, Fig. 1H, as previously described [18] and the maleylated tryptic fragment, derived from a chymotryptic-like cleavage at tryptophan-172, is obtained from fraction 1, Fig. 1G. Results of ordinary analyses, and consistent support for the structures determined, are evident from Tables 2 and 3 but further comment is given below for peptides, residues or overlapping regions presenting special problems.

Overlapping Regions Most of the expected peptides were obtained pure from each digest, as seen in Table 2, and interconnecting fragments are therefore generally abundant. Short or comparatively few overlapping peptides, however, are in the regions between the tryptic peptides T1/T3 (via T2), T5/T8 (via T7), T8/T9 and at a few places within the largest tryptic peptide T13. The structures of T2 and T7 were deduced from analyses of several overlapping fragments (Table 2). Although these two tryptic peptides were never obtained completely pure, their sequential degradations (Table 2) support the structures deduced. Therefore,

429

the difficulty in purification of T2 and T7 probably has no significance but might be of interest in regard to possible microheterogeneities (below). The two tryptic peptides T8 and T9 are overlapped by C10 and M1 (Table 2). This overlap is short and involves an unexpected chymotryptic cleavage of a Thr-Lys bond. Cleavage of this bond is, however, supported not only by recovery of the overlapping peptide (CIO) but also of the preceding chymotryptic peptide (C9). Furthermore, cleavage in high yield at a single threonyl bond has been reported in horse liver alcohol dehydrogenase, at threonine-82 [33]. Finally, the chymotryptic peptide C10 cannot be better placed anywhere else, and this overlap, as well as all the others in the structure, is consistent with the recovery of the large maleylated peptide 1-172 (Table 4). Problems with establishing some of the overlapping regions within T13 are associated with particular positions and deamidations (below).

Individual Positions No free N-terminal residue can be detected in the intact protein, suggesting a blocked N-terminus [35, 14,2]. This was recovered in peptides T1, C1 and P1 (Table 2). Since they could not be degraded by the Edman method, T1 was cleaved by partial acid hydrolysis in 9.7 M HCl for 30 h at room temperature. This produced three fragments accounting for all original amino acids. One fragment was acidic (SerIle-Pro-Glu, T 1 HCl1, Table 2), one neutral (Thr-GluLys, T1 HCl2, Table 2) and one basic (Thr-Gln-Lys, T 1 HC1 3, Table 2). The latter two peptides are derived from the same C-terminal part of T1 due to partial deamidation of a Thr-Gln-Lys sequence, in agreement with the original charges of T1 and C1 (Table 3). These results establish that the N-terminus of the protein is an acylated serine residue, which is also a common feature of many other proteins [36]. Repetitive proline residues at positions 24- 28 complicate analyses by the dansyl-Edman method. Separate isolations and complete degradations of all the tryptic peptides T4, T5 and T6, however, fully prove the structure deduced from the chymotryptic peptides covering this region. T4, T5 and T6 were obtained from the tryptic digests of both the whole protein and the isolated chymotryptic peptide C 3, showing that the Lys27-Pro28 bond is quite susceptible to hydrolysis by trypsin.

Fig. 1. Fractionations on Sephadex G-50 ofpeptide mixtures from [‘4CJcarboxymethylated yeast alcoholdehydrogenase. Proteolytic agents used were trypsin (A), chymotrypsin (B), pepsin (C), a staphylococcal protease (D: only soluble peptides. E : redigestion of precipitate with a Myxobacter protease), a Myxobacter protease (F),Trypsin on maleylated substrate (G), and CNBr (H). Radioactivities ( __ ) were measured a spectrophotometer (280 nm) or a Uvicord flow cell (254 nm). Column dimenin a gas-flow counter and absorbances (1-cm path; -----)in sions, 2.5 x 100 cm in (B) and (E), 5 x 100 cm in all the other cases. Elution with 0.1 M ammonium bicarbonate

Table 2. Positions and sequence analyses ofpepiides from an N-ierrninai 172-residue segment of carboxymethylated yeast alcohol dehydrogenase -shows a residue analyzed by sequential degradation with the dansyl-Edman method, (-) indicates low recovery, dindicates a residue proven to be C-terminal in a peptide by recovery at this stage even without hydrolysis. Peptides that are underlined denote those for which complete data are given in Table 3 or, for P16,PI7 and B3T3, in [38]. Nomenclature of peptides is explained in the Methods section. Figures within parentheses after names of all major peptides give the fraction in Fig. 1 from which the peptide was purified

.

~

- -

Alanine

1.0(1)

ne

iroleucine

LeUCl

a

24

None

7

~

11

None

-

Tyr

22

1.8(2)

Tyr

13

0.7(1)

- -

. .

- -

1.111)

12

29

0.29

C7

-

- - -

- -

n.g(i)

1.8(2)

- -

~

l.O(l)

1.0(1) . .

-

1.8(2) 1.3 ( 1 ) 2.1 ( 2 ) 1.1 ( 1 ) 1.0(1) -

-

i.o(i)

i.n(i)

o . n ( i ) o . R ( ~ )0 . 8 ( 1 ) 2.1 ( 2 ) 2.1 ( 2 ) 1 . 1 ( 1 ) 1.1(1) 2 0 ( 7 ) l,O(l)

19

-0.09

18

- . . . o . ~ ( I )1.1(1) - -

-

-

1.1(1)

-

-

-

- . . -

- -

1.0(1)

Asp

8

~

I . S ( Z ) ~ ~ , O ( II .)O ( I ) 0 . 9 ( 1 ) - . 2.2(2)

-

0.7(1)d1,1(1) . . .

-

1 . 0 ( 1 ) 3 . 2 ( 3 ) 2 . 8 ( 3 ) 2.9(3) 1.2(1) 1.2(1) 0.4 1.2(1) 1.0(1)

-

1.1 (1)

- .

7

0

0

-0.25

-0.20

9

P4

RM3

T5

R3

Leu

5 Leu

14 Tyr

21

Ser

R

- - 2.0(2) 2 0 ( 2 ) 1.9(7) l . O ( I ) - - 0.9(1) 0 . 8 ( 1 ) - 0.9(1) - . - . - . - . . - . . . - - - - - - - - - - - - + (2)f - 0 . 9 ( 1 ) 0.9(1) 3 . 9 ( 4 ) 2 . 7 ( 3 ) 2 . 2 ( 2 ) - - o.g(i) o.g(i) - - - - - 0 . 8 ( 1 ) 2 . 6 ( 3 ) O.S(l) - - - 0.9 ( 1 ) - -

- - . -

. .

8

-0.14

P2

- . - - . . . . - - 2 . 2 ( 2 ) 2.1 ( 2 ) 1 . 1 ( 1 ) 1.0(1) - - - - 1.1(1) 1.2(1) - - 2.1 ( 2 ) 3.1 ( 3 ) 1 . 1 ( 1 ) - -

14

020

C1

~

-

- -

27

0

19

1.9 (2)

- . -

-

2.2 ( 2 )

-

-

1.1 (1)

- -

12

0

C11

-

1.2 ( 1 )

1 . 2 (1)

- -

0.8(1) 2.8 ( 3 )

11

Wis

9

o.g(i) 0.7(1)

Lys

4

- -

i.n(i)

~

LYS

24

Leu

21

z.i(z) i.o(i) 1.2(1) 0 8 ( l )

Val

20 Gly

23

- -

1.1(1) 3.2(3) 0.9(1)

1.0 (1)

-

Gly

3

- -

o.g(i)

+

.

LYS

5

- -

i.o(i)

- -

- -

I.O(I)

- - - -

lle

8

- -

o.g(i)

- -

l.O(l) .

- -

Ala

5

- -

o.g(i)

0.3 ( 1 )

- - - .

0.9(1)

- - -

Z.O(Z)

1.0(1)

- - -

1 . 2 (1)

1.1(1)

-

.

-

2.0 ( 2 )

-

16

-

.

. .

-

C13 -0.42

- - -

-

- -

l.O(l) . . . (1)

- - -

-

- - - -

1.0 ( 1 )

- -

47

0

ill

1.0(1) l l ( 1 )

- - -

- -

26

0

C12

- - - - - - -

35

-0.48

Peptlde 110

O/O.llb

82

l.O(l) 0.8(1) 7 . 0 ( 7 ) 6.0 (6) 6.8 ( 7 ) 4.9 ( 5 ) 1.2(1) 1.2(1) 1.2(1) 1.2(1)

2.2 ( 2 )

~

- -

1.2 ( 1 ) 1 . 1 ( 1 )

9

-0.11

Ml

- - - 3.9(5)d3.7(5)d3.9(5)d1.2(1) . . - . 0.9 ( 1 ) 0 9 ( 1 ) 0 . 8 ( 1 ) t e ( 1 ) - - - - - - - - - IR(Z) 1 . 0 ( 1 ) 2 . 1 ( 2 ) 2.0(2) 1 9 ( 2 ) - 1.1(1) - - - - - - . - - - 0.8(1) . . . . . . . - . . . . + (1) - + (1) - + ( 1 ) + (2)f -

- -

-

-

- - - - -

~

13

-0.45

ClO

l . 8 ( 2 ) 0.911) 1.2 ( 1 )

-

1.1 ( 1 ) 1.0(1)

- -

(26)

-0.15

TRC3

-

C18

-

1.6(2) 2.9(3)

lR

0.73

P9 R9

13

Lev

12

- - - -

CytiCn)

14

0.8(1)

. .

- - -

- -

~

~

TYT

24

lS(2)

-

1.7(2) 0.8jl)

1.7(2)

1.9(2)

- 1.0(1) 0.8(1) . .

9

0.48

CZO'

R12

8

0.27

18

0

T13Hll

Leu

23

Leu

-

4 Thr

9

1.1 ( 1 )

Tvr

20

1.2 (1)

Ala

8

1.0 ( 1 )

- - 5 . 8 ( 6 ) 1.2 ( 1 ) - - l.1(1) - . . . - - . . - . - - - 0 . 9 ( 1 ) 0.8 ( I ) 1.6(2) 1.0(1) - -_ 0.9(1) - 0.9(1) 0.8(1) 0.7(1) 0.7(1) - 0.711) - - 0.8(1) - . - - . - - - . . . . - - - . - - - - - 1.7(2) - 0.9(1) 0.8(1) 0.9(1) - -

. -

1.2(1) 1.0(1) 2.8 ( 3 ) 2.9 ( 3 ) 1 . 3 ( 1 ) 1.3(1) 2.1(2) -

0.7(1)

1.1 ( 1 ) LO(1)

0.9(1)

- - - - . - - - - .

- -

1.1 ( 1 ) 1.2(1)

- -

6

0

R10

- - - - - - - . - - l O ( 1 ) 2 . 3 ( 2 ) 1.0(1) - . 0.8(1) 1 . 9 ( 2 ) 0 . 9 ( 1 ) 1.1(1) 1.1(1) - - - - 1 . 9 ( 2 ) 3.0(3) l O ( 1 ) - - - - 1.2il) Ll(1)

0.40/0.57c

1.6(2) O.R(l) 3.8(4) 3 . 7 ( 6 ) 1.0(1) 1.2(1)

76

0.54

1 . 1 ( 1 ) 0.9(1) 3 . 0 ( 3 ) 2.2(2) 2.1 ( 2 ) 2.1 ( 2 ) 2.2 ( 1 ) 3.1 ( 3 )

-

2.5(3) 1.1(1)

8

1.05

T13H1

Treated with CNBr during purification, explaining deamidations. Two forms, depending on C-terminal Hsl or Hse, respectively. Both forms deamidated. Two forms with different charges, from deamidations. Sequence analyses show that C1 contains a Val-Ile bond, and that M1, C11 and T9 contain Val-Val-Val, explaining the low recoveries of Val and Ile. Present as Hsl and Hse. From tryptophan-containing peptides obtained after redigestion with trypsin (B2) or chymotrypsin (T8).

N-temrnun

Sum

Lyrlne Histidine

Phenylalanine Tryptophan

Tyrosine

.

-

Vallne Neth,onlne

. -

. -

0.9(1)

12(1) 1.9 ( 2 )

l.O(1)

~

-

18

0.32

T1

Proline Glycine

Threonine Serine Glutimcacid

Aspartic a c i d

Cm-cysterne

Compoii t i o n

Recovery (PI)

MobilityatpH6.5

Charactenstic

-

-

IPU

3

- - - -

0.8(1)

.

2.0 ( 2 )

1.1(1) 2.1 ( 2 )

0.9 (1)

- - -

116

-

0.9 ( 1 )

- . - . - - - -

43

-0.51

C25

-

0,8 (1)

- -

1,2(l) 1.2 ( 1 )

- - -

.

-

- . -

18

-0.35

val

10

- - -

0.8(1)

-

,$la

3

1.0(1)

P L W

MO+

5

- - - - - - - . . - . 0.3 (1) . - . 1.1(1) - - - - - - 0.8 (1)

-

1.0(1)

-

07(1)

6

0.27

RM7

. . . . . - - 0.8 ( 1 ) - -

-

1.0 ( 1 )

- - -

11(l)

.

- - - -

19

0.54

T13H12

Table 3. Data for peptides establishing overlapping regions in the N-terminal half of carboxymethylated yeast alcohol dehydrogenase Values are molar ratios (those below 0.3 omitted) without corrections for destruction, incomplete hydrolysis or impurities. Nomenclature of peptides is given in the Methods section. Peptides listed are underlined in Table 2. and ordered according to their positions in that table

1

Glutamic a c i d

a

Hsl present.

t0.2

1

1.1

Phenylalanine

A r g i n i ne

1

3.5

Tyrosine

2.5

4

5.1

Leucine

H i s t i d i ne

5

2.2

I s 0 1 eucine