Effect of secondary interaction on the enzymatic activity of trypsin-like enzymes from Streptomyces

ARCHIVES

OF

BIOCHEMISTRY

Effect

AND

BIOPHYSICS

of Secondary Trypsin-like KAZUYUKI

Shionogi

Research Laboratory,

166, 764-771

Interaction

(1973)

on the

Enzymes

from

MORIHARA

Enzymatic

Shionogi

and Co., Ltd.,

Received

January

of

Streptomyces TATSUSHI

AND

Activity

Fukushima-ku,

OKA Osaka, 665 Japan

10, 19’73

The effect of secondary interaction with substrate on the enzymatic activity of trypsin-like enzymes from Streptomyces was studied using Z-Lys-(Ala),, Z-(Ala),Lys-OMe, Z-Lys-X-Ala, and Z-X-Lys-OMe (m = 14; n = (t2; X = various amino acid residues) as substrates, and a comparison was made with bovine trypsin. These peptides are susceptible to cleavage at the peptide or ester bonds containing the carbony1 group of L-lysine, which enabled determination of the effect of chain-length on either side of the sensitive L-lysine residue in the first two types of peptide, and the effect of side-chains of the amino acid residues immediately neighboring on either side of the sensitive L-lysine residue in the latter two types of peptide. The results indicate that the enzymatic activity of the trypsin-like enzymes are little affected by secondary interaction, similarly as seen with bovine trypsin.

The specificity of certain proteinases has been shown to be determined not only by the adjacent amino acid residue(s) at either or both sides of the splitting point in peptide substrates but also by amino acid residues more distant from the catalytic point. The former specificity may be called “primary specificity,” and the latter effect “secondary interaction” (1). Generally speaking, it is likely that the activity of proteinases which show broad specificity is greatly determined by secondary interaction, as seen in papain (a), pepsin (l), subtilisins (3, 4), etc. To examine whether the above observations are general, the effect of secondary interaction on the enzymatic activity of trypsin-like enzymes from Streptomyces (5-7) was determined, and a comparison was made with bovine trypsin. These enzymes show most stringent or narrow specificity against basic amino acid residues at the carbonyl side of the splitting point, and are inhibited by specific inhibitors such as DF’P,’ TLCK, and soybean trypsin inhibitor. 1 Abbreviations: fluoridate; TLCK, ketone.

The effect of the length of chain on either side of the splitting point was determined using Z-Lys-(Ala),, and Z-(Ala),-Lys-OMe (m = 14; n = O-2) as substrates. The effect of the kinds of amino acid residue at either side of the specific basic amino acid residue in peptide substrates was studied using Z-Lys-X-Ala and Z-X-Lys-OMe (X = various amino acid residues) as substrates. MATERIALS

DFP, diisopropyl phosphotosyl-L-lysine chloromethyl 764

Copyright All rights

@I 1973 by Academic Press, of repmduction in any form

Inc. reserved.

AND

METHODS

Enzymes. Crystalline bovine trypsin (twice recrystallized) was obtained from Worthington Biochemical Corporation, New Jersey. Highly purified trypsin-like enzymes from Streptomyces griseus (6), and Streptomyces erylhreus (7) were kindly donated by Dr. Y. Narahashi of Physico-Chemical Research Institute (Saitama), and Dr. N. Yoshida of this laboratory, respectively. A trypsin-like enzyme from Streptomyces fradiae was purified according to the method described previously (5,7). These enzyme preparations appeared homogeneous when examined by disc-electrophoresis at pH 8. Sub&ales. Z-Lys-(Ala), (m = l-4), and Z-LysX-Ala (X = glycine, D and L-leucine, and L-phenylalanine) were synthesized according to the method described in another paper (8). The other peptides were synthesized as described below. Abbreviated designations of amino acid deriva-

SPECIFICITY

OF TRYPSIN-LIKE

tives, peptides or their derivatives conform to the tentative rules of the IUPAC-IUB Commission on Biochemical Nomenclature. Except when specified, the constituent amino acids were all of the L-configuration. Deferminufion of peptidase activity. Due to the low solubility of some of the peptide substrates, dimethylformamide was included in the reaction mixture. The reaction mixture (l-10 ml) containing 0.05 M Tris-buffer (pH 8.2), lOc/, dimethylformamide, an appropriate concentration of peptide, and a suitable amount of enzyme was kept at -10”. At suitable time intervals, appropriate amounts of aliquots (0.1-1.0 ml) were transferred to a test tube containing 1 ml of citrate buffer (0.5 BL, pH 5.0) and kept in an ice-water bath to stop further hydrolysis until all the samples had been of hydrolysis was measured by taken. The extent the ninhydrin method of Yemm and Cocking (9). The colonr yields by the ninhydrin method of (+ly-Ala, Ala-Ala, Leu-Ala, Phe-Ala, Ala-Ala-Ala, and Ala-Ala-Ala-Ala, based on L-leucine as lOOTi, were 93, 133, 88, 110, 78, and 83’;;, respectively. Z-Peptide substrates containing L-lysine used in this study or their Z-amino acid or Z-peptide fragments released by trypsin action gave considerably ninhydrin colour owing to the e-amino group of lysine in the respective derivatives; their color yields were in the range of 30-400; based on L-leutine as lOO(;; . Therefore, caution was required in t,he determination of the increase of the ninhydrin value by the enzymatic action of these peptides. The sites of action of enzymes upon substrates were determined by paper chromatography of the hydrolyzates in comparison to authentic compounds, or by paper electrophoresis at pH 1.9 (0.6 N formic acid and 2 N acetic acid, 1: 1, by volume). Determination of eslerase c&i&y. Esterase act ivity was determined with the aid of a Radiometer t.ype TTTl pH-stat equipped with a syringe burette, a type SBR2c recorder, and a thermostatically controlled reaction vessel (30”). The reaction was carried out in 0.1 M KC1 at pH 7.5, and 0.05 N NaOH was used as titrant. Kinetic study. In all cases, satisfactory Michaelis-Menten kinetics were observed, and plots of l/v vs l/S (Lineweaver-Burk plots) permitted the fitting of definite straight lines. For each determination of K, and V derived from such plots, initial rates were measured from five (or more) values of the initial substrate concentrations S. lIepending upon the rate of enzymatic cleavage, t,he enzyme concentration was suitably adjusted. In the calculation of k,,t, the molecular weight of t,rypsin was assumed to be 24,000 (lo), and those of the trypsin-like enzymes from Strepfomyces were 20,000 (7).

ENZYMES Synthesis

765 of Peptides

or-Z-(e-BOC-)Lys-O~~e.2 a-Z-(e-BOC-)Lys (3.99 mM), obtained from the dicyclohexylamine salt as described in another paper (8), was treated with CH?Nz in the usual manner (ll), with ether (80 ml) as the solvent. After concentration in z’ucuo, the product was obtained as an oily syrup. Yield, 1ooc/; . a-Z-Lys-OMe-HCZ. The above ester (3.8 mM) was treated as usual (12) with N-HCl in acetic acid (20 ml). A syrup was obtained as the product. Yield, 1007;. E-BOC-Lys-OMe-HCZ. The product was obtained by catalytic hydrogenolysis of CX-Z-(e-BOC-) Lys-OMe (18.4 mM) in the usual manner (11). Crystallized from ether. Yield, 97.6c;; mp 153154”. Z-Gly-(&OC-)Lys-OMe. Z-Gly-ONP (3.52 mu) and E-BOC-Lys-OMe, prepared by neutralization of the hydrochloride (3.57 mM) with cold triethylamine, were coupled in the usual manner cl.?), with CH&12 (30 ml) as the solvent. An oily syrup was obtained as the product. Yield, 90’;(. Z-Gly-Lys-OMe-HCZ. The above ester (3.17 nm) was treated with N-HCl in acetic acid (20 ml). An oily syrup was obtained as the product. Yield, 94.30; . Z-Ala-(6.BOG’-)Lys-OMe. Z-Ala-ONP (2.52 mu) and e-BOC-Lys-OMe (2.48 rnM) were coupled, with CH&lz (20 ml) as the solvent. The product was purified by column chromatography on silicagel using benzene-ethyl acetate as the effluent. Crystallized from ether-petroleum ether. Yield, 83.8yo ; mp 89-90”. Z-Ala-Lys-OMe-HCZ. The above ester (2.65 mM) was treated with N-HCl in acetic acid (15 ml). An oily syrup was obtained as the product. Yield, 97yc. Z-o-Ala-(E-BOC-)Lys-OMe. Z-D-Ala-ON (3.43 mM) and c-BOC-Lys-OMe (3.52 mM) were coupled, with CH#& (30 ml) as the solvent. The product was crystallized fromether-petroleum ether. Yield, 45.17;; mp 90-92”. Z-o-Ala-Lys-OMe-HCI. The above ester (1.50 mM) was treated with N-HCl in acetic acid (20 ml). The product was crystallized from ethyl acetate. Yield, 90.5yc; mp 90-92”. Z-Leu-(e-BOC-)Lys-OMe. Z-Leu-ONP (3.56 mM) and e-BOC-Lys-OMe (3.59 mM) were coupled, with CH&lz (30 ml) as the solvent. An oily syrup was obtained as the product. Yield, 90.77;. Z-Leu-Lys-OMe-HCl. The above ester (3.15 mM) was treated with N-HCl in acetic acid (20 ml). 2 The data on elemental for editorial review.

analysis

were presented

766

MORIHARA

An oily syrup was obtained as the product. Yield, 82.1%. Z-Phe-(c-BOG-)Lys-OMe. Z-Phe-ONP (3.59 mM) and s-BOC-Lys-OMe (3.61 mrvr) were coupled, with CH&lz (30 ml) as the solvent. An oily syrup was obtained as the product. Yield, S4.1y0. Z-Phe-Lys-OMe. The above ester (2.95 mM) was treated with N-HCl in acetic acid (20 ml). The product was crystallized from ether-petroleum ether. Yield, 77.3y0; mp 193.5195”. Z-Ala-Ala-(&OC-)Lys-OMe. Z-Ala-ONP (1.33 mM) and Ala-(e-BOC-)Lys-OMe, prepared by catalytic hydrogenolysis of the corresponding Z-peptide (1.27 mM), were coupled, with CHzClz (20 ml) as the solvent. The product was purified by column chromatography on silica gel using benzene-ethyl acetate as the effluent. The product was crystallized from ether. Yield, 74.1%; mp 129-130°. Z-Ala-Ala-Lys-OMe-HCl. The above ester (2.26 mm) was treated with N-HCl in acetic acid (12 ml). The product was crystallized from etherpetroleum ether. Yield, 97.90/,; mp 96-98”. RESULTS

The effects of the length of chain from the point of cleavage to the C- and N-termini on the hydrolysis of peptide substrates by trypsin-like enzymes from Streptomyces and bovine trypsin were determined using Z-Lys-(Ala), and Z-(Ala),-Lys-OMe (m = l-4; n = O-2) as substrates. The kinetic studies of the four enzymes against these peptides are summarized in Tables I and KINETIC

AND OKA

II. For convenience, the amino acid residues in a peptide substrate are numbered P1, Pz, etc. to the left of the bond hydrolyzed and PI’, P2/, etc. to the right. Table I indicates that Z-Lys-Ala is negligibly hydrolyzed by these enzymes. Since Z-Lys-OMe is a good substrate for all these enzymes (Table II), this might be ascribed to the presence of a negatively charged group at PI’. Z-Lys-(Ala)p is hydrolyzed considerT ably by these enzymes. The elongation of the peptide chain with n-alanine from Pz’ to Pa’ results in a further increase of hydrolysis by each of these enzymes as seen by comparison of Z-Lyst(Ala)z and Z-Lys;(Ala), , but further elongation from Pal to P4’ does not. A similar result has previously been obtained with trypsin by Yamamoto and Izumiya (14); they determined the effect of chain-length using (Gly)z-Lys;(Gly),, (m = 14) as substrates, and found that the effect of m on hydrolysis was in the order 3 > 4 > 2 >> 1. Table II would indicate that elongation of the peptide chain from the bond hydrolyzed to the N-terminus in peptide substrates does not increase the esterase action of the four enzymes. The study of Yamamoto and Izumiya (14) with trypsin has indicated that both (G~Y)~-L~s~NH~ and (G~Y)~-L~s~NH~

TABLE I PARAMETERS FOR HYDROLYSIS OF Z-Lys-(Ala),

-

Peptides

St. griseus Km

i

hat

(maa) (set-I)

St. fradiae

!

-

BY TRYPSIN-LIKE

-

La/ Km KG78 (mM) -__~ 0.0

--

Z-Lys-Ala Z-Lys-(Ala)2 t

3.3 4.1 * 0.2 * 0.1

1.2

Z-Lys-(Ala)a T

2.2 * 0.2 f

8.5 0.3

3.9

Z-Lys-(Ala), T

6.4 23.7 + 1.6 f 5.2

3.7

5.2 15.2 0.4 f 0.7

2.9

f

1.6 15.4 0.1 f 0.3

9.6

f

1.2 0.5 f I

6.9

f I

8.3 1.0

Trypsin

St. erythreus

I

ENZYMM”

kest (set-I)

I

KWb k oat (set-I) 0-f)

p/ m

0.0

3.4 0.6 f

1.4 0.1

10.7 5.7 k 2.8 * 1.0

0.5

f

3.5 0.7 f

7.2 1.0

3.7 4.6 k 0.4 f 0.4

1.2

f

5.7 12.0 1.9 f 3.2

3.3 k 0.1 f

0.7

f

2.4 0.1

-

a The reaction mixture contained 0.05 M Tris-buffer (pH 8.2), 10% dimethylformamide, and a suitable amount of enzyme. Substrate concentrations used were 1.5-4 mM. The reaction was carried out at 40”. The arrows show the bonds split.

activity

55

1650

500

890

in t,he presence

60 iz2

f2

0.11 0.01

0.036 0.002

f2

46

k oat (set-I)

0.055 0.007

KWZ bM)

II

f2

fl

0.067 0.004 0.037 0.002

34

61

57 zk8

k 03t (see-I)

St. jradiae

-

920

910

f

f

0.068 0.002

0.084 0.003

0.10 0.01

1150 f

K, bM)

&l

fl

fl

2“

25

31

-

f

f

f

0.079 0.009

68

0.039 0.003

mM.

106 z?z6

f2

101 +3

k cat (see-I) 0.23 0.02

LL bM)

Trypsin

used were 0.03-0.5

320

300

310

kcat/Km

-

&ZYMI~.Y?

roncent,rations

I-

k cat (set-I)

2. erytlwefrs

ny TRYPSIN-LIKE

kest/Km

OF Z-(Ala),-Lys-ONe

TABLE

of 0.1 M KC1 at pH 7.5 and 30”. Substrate

f

f

f

0.049 0.002

&I cm4

PARAMETERS FOIL HYI)HOLYSIS

was determined

f

Z-(Ala),-Lys-OMe T

a The e&erase

f

f

-0Me t

Z-Ala-Lys

Z-Lys-OMe T

Peptides

&NE:TIC

1350

1750

440

LtlKm

*

f

0.91 0.05

1.8

1.2

f

f

f

0.64 0.04

2.0 0.3

5.2 0.4

6.3 0.9

f

f

f

f

1.15 0.03

4.8 0.5

15.2 0.7

3.1 0.3

1.8

0.0

2.4

2.9

0.5

cd&

a The reaction mixture contained 0.05 M Tris-buffer (pH 8.2), 1O70 dimethylformamide, used were 1.5-7 mM. The reaction was carried out at 40”.

1.3 0.1

f

3.1 0.2

4.1 0.1

0.7

f

1.7 0.2

f

0.3

Z-Lys-Phe-Ala t

f

Z-Lys-Leu-Ala T

3.3 0.2

f

1.8 0.4

k cat (set-l)

&I8 (m@

k Cat (set+)

k,,.tlK,

St. jradiae

St. griseus

0.0

f

Z-Lys-Ala-Ala t

5.3 1.7

KWl b-d

III

OF Z-Lys-X-Ala

TABLE PARAMETERS FOR HYDROLYSIS

Z-Lys-D-Leu-Ala

f

Z-Lys-Gly-Ala t

Peptides

KINETIC

--

n

2.4 0.5

2.4 0.3

3.4 0.6

1.4 0.J

0.63 0.12

k cat (set-1)

f

amount

0.96 0.13

0.64 zk 0.06

f

f

and a suitable

i

f

f

f

7.9 2.3

&I bM)

f

f

f

f

f

f

f

0.2

0.0

0.2

0.5

0.1

---EC

,,,tlK,

concentrations

0.92 0.08

0.96 0.04

5.7 1.0

0.97 0.23

k cat (set-l)

Trypsin

Substrate

4.1 0.5

5.0 0.4

10.7 2.8

13.1 f 4.1

of enzyme.

0.4

0.0

0.3

0.4

0.1

ENZYMES"

St. erythreus

BY TRYPSIN-LIKE

P

s IJ 0

E

g El

1 The e&erase

activity

0.016 0.001 fl

fl

f

0.093 0.01

0.044 0.002

f2

fl

2400

910

58

500

830

-

T

f2

f3

19.8 zk 0.4

zt2

60

3.8 0.1

61

84

k cat (set-I)

St. fradiae

f

IV

1980

690

47

910

1250

OF Z-X-Lys-OMe

TABLE

f

f

f

f

0.043 0.001

0.046 0.003

0.084 0.003

0.14 0.01

Kl8 (md

16.6 0.2

570

320

0.054 zt 0.004

46

22.4 0.2

2.7 0.2

68

84

mM.

f2

f

f

0.14 0.02 0.016 0.001

f2

f2

k oat (set-I)

0.039 zko.003

0.11 0.01

Km (md

Trypsin

__concentrations used are 0.03-0.5

f

f

f

f

13.6 0.1

59

300

300

f

25

42

i

ENZYMICS”

2.7 **0.1

fl

fl

k oat (set-9

St. erythreus

BY TRYPSIN-LIKE

0.029 f 0.001 -___ of 0.1 M KC1 at pH 7.5 and 30”; substrate

0.087 0.007

0.081 0.005

0.067 0.004

0.067 0.006

0.010 rt 0.001

f

f

f

f

Km bMd)

FOR HYDROLYSIS

in the presence

39

40

5.4 0.3

55

62

k oat (set-*)

0.11 0.01

0.075 0.002

K, (4

PARAMFSTRRS

St. griseus

was determined

f

Z-Phe-Lys-OMe T

f

f

t

f

f

Z-Leu-Lys-OMe 5

z-D-Ala-Lys-OMe

Z-Ala-Lys-OMe T

Z-Gly-Lys-OMe A

Peptides

KINETIC

770

MORIHARA

are hydrolyzed approximately 20 times faster than Gly-LysrNHz. The low rate of hydrolysis of Gly-Lys-NH2 could therefore be ascribed to the presence of a positively charged group at Pz. The effect of the amino acid residue on either side of the sensitive L-lysine residue was examined using Z-Lys-X-Ala and Z-XLys-OMe (X = various amino acid residues) as substrates. The kinetics of the hydrolysis of Z-Lys-X-Ala by the trypsin-like enzymes and try&in are summarized in Table III, and indicates that the four enzymes show a stringent stereo-specificity with respect to X (at PI’). The effect of the nature of the side-chain at this position is also considerable; the proteolytic coefficient (k&K,) of the peptides containing amino acids such as L-ala&e, L-leucine, or L-phenylalanine is several times higher than that of the peptide containing glycine as X. In the hydrolysis of Z-X-LyspMe (Table IV), considerable stereo-specificity is also observed for X, while the effect of the nature of the side-chain at Pz is small. DISCUSSION

The present study indicates that the effect of secondary interaction on the enzymatic activity of trypsin-like enzymes from Streptomyces and trypsin is very small in comparison with that for the other serine proteinases. For example, the esterase activity (k,,t/K,) with Z-(Ala)z-Lys-OMe as substrate is only two or three times that of Z-Lys-OMe as shown in this paper. In contrast, with pancreatic elastase (15) and subtilisin (16, 17), it has been shown that the esterase activity on Ac-(Ala)P-OMe is more than 1000 times higher than that on Ac-Ala-OMe. Even with cr-chymotrypsin, the esterase activity on Ac-(Ala)z-Phe-OMe has been shown (unpublished data) to be about 35 times higher than that on Ac-Phe-OMe. It has been shown (18) that the backbone chain involving the CO of Ser 214 and the NH and CO of Gly 216 in r-chymotrypsin is implicated in hydrogen bonding in an antiparallel P-pleated arrangement with tripeptide chloromethyl ketone. A similar situation has also been observed (19) with

AND OKA

subtilisin BPN’, the corresponding backbone chain being Ser 125 and Glv 127. This mode of binding offers the possibility of hydrogen bonding between enzyme and substrate. Therefore, the increased susceptibility of the Ac-tripeptide esters to hydrolysis by 01chymotrypsin and subtilisin (and possibly that to pancreatic elastase) as mentioned above would be correlated with the ability to form three hydrogen bonds. The question arises whether such hydrogen bonding is not operative in hydrolysis with the trypsin-like enzymes or trypsin. The model complex between bovine trypsin and pancreatic trypsin inhibitor suggests (20) that two hydrogen bonds of the antiparallel pleated sheet type may form between the CO of Ser 214 on the enzyme and the NH of Lys 15 on the inhibitor, and between the NH of Gly 216 on the enzyme and the CO of Pro 13 on the inhibitor. Therefore, it may be possible to consider that although the same hydrogen bonding is formed between trypsin and tripeptide substrates as between chymotrypsin and tripeptides, their cont,ribution to hydrolysis may be small because of the large contribution of the specific binding pocket which enfolds the basic amino acid residues. Since it has been shown (21) that all the serine enzymes described above have similar and yet unique binding pockets (these are especially alike in trypsin and r-chymotrypsin), the Asp 189 residue at the bottom of the pocket in trypsin must play an important role (22), this amino acid residue being replaced with Ser in y-chymotrypsin. It has also been found (23) that the pocket of trypsin is somewhat deeper than that of y-chymotrypsin. Jurasek et al. (24) have found that the amino acid sequences around the active serine and histidine in the trypsin-like enzyme from St. griseus (identical with the enzyme presented here) arc remarkably similar to the corresponding ones of bovine trypsin. A comparison of the sequences of the internal regions strongly suggests that both the enzymes have a very similar conformation. Although the amino acid sequences of the other two Streptmyces enzymes have not yet been determined, it might be considered that the effect of second-

SPECIFICITY

OF TRYPSIN-LIKE

ary interaction observed with these enzymes is negligible for the same reason as proposed for trypsin.

ACKNOWLEDGMENTS We are greatly indebted to Dr. Y. Narahashi of Physico-Chemical Research Institute (Saitama), and Dr. N. Yoshida of this laboratory, for their supply of purified enzyme preparations of St. griseus and Sf. erythreus, respectively. REFERENCES 1. FRUTON, J. S. (1970) Adv. Enzymol. 33, 401. 2. SCHIXHTER, I., .~ND BERGER, A. (1967) Biothem. Biophys.

Res. Commun.

27, 157.

K. OKA T., SND TSUZUKI, H. (1970) Arch. Biochem. Biophys. 138,515. 4. MORIHARA, K., TSUZUKI, H., AND OKA, T. (1971) Biochem. Biophys. Res. Commun. 42,lOOO. 5. MORIHARA, K., AND TSUZUKI, H. (1968) Arch. 3. MORIWARA,

Biochem.

Biophys. 126,971. Y., AND FUKUNAGA, J. (1969) J. Biochem. (Tokyo) 66,743. YOSHIDA, N., S~s.4~1, A., AND INOUE, H. (1971) Fed. Eur. Biochem. Sot. Left. 16,129. MORIHARA, K., AND OKA, T. (1973) Arch. Biochem. Biophys. in press. Y~MM, E. W., END COCKING, E. C. (1955)

6. NARAHASHI, 7#. 8. 0.

Analyst 80, 209. 10. W.~LSH, K. A. (1970) in Methods in Enzymology (Pearlmann, G. E., and Lorand, L., eds.), Vol. XIX, p. 41, Academic Press, New York.

ENZYMES

11. SCHWYZER,R., AND RITTEL,

W. (1961) H&r.

Chim. Acta 44, 159. 12. SCHNABEL, E., KLOSTERMEYER, BERNDT, H. (1971) Liebigs Ann.

H., AND Chem. 749,

90.

13. BODANSZKY,)M.

(1%5) Xafure

(Lo&m)

175,

685. 14. YAMAMOTO, T., AND IZUMIYA, N. (1967) ~mh. Biochem.

Biophys.

120, 497.

15. GERTLER, A., AND HOFMANN, T. (1970) Can. J. Biochem.

46, 384.

16. GERTLER, A. (1971) Eur. J. Biochem. 23,36. 17. MORIHARA, K., AND TSUZUKI, H. (1969) Arch. Biochem. Biophys. 129, 620. 18. SEGAL, D. M., POWERS, J. C., COHEN, G. H., DAVIES, D. R., AND WILCOX, P. E. (1971) Biochemistry 10, 3728. 19. ROBERTUS, J. D., ALDEN, R. A., BIRKTOFT, J. J., KRAUT, J., POWERS, J. C., AND WILCOX, P. E. (1972) Biochemistry 11, 2439. 20. BLOW, D. M., WRIGHT, C. S., KUKLA, D., R~~HLMANN, A., STEIGEMANN, W., AND HUBER R. (1972) J. MOE.Biol. 69,137. 21. SHOTTON, D. M., AND WATSON, H. C. (1970) Nature (London) 226, 811. 22. STEITZ, T. A., HRNDRRSON, R., AND BLOW, D. M. (1969) J. Mol. Biol. 46,337. 23. STROUD, R. M., KAY, L. M., AND DICKERSON, R. E. (1971) in Symposia on Quantitative Biology, Vol. XXXVI, p. 125. 24. JURASEK, L., FACKRI:, D., AND SMILLIE, L. B. (1969)

37,99.

Biochem.

Biophys.

Res.

Commun.