Comparison of Streptomyces griseus and bovine trypsin by active site analysis using fluorescent acyl groups

Biochimica et Biophysica Acta 913 (1987) 292-299

292

Elsevier BBA 32846

Comparison of Streptomycesgdsem and bovine trypsin by active site analysis using fluorescent acyi groups Kazutaka Tanizawa, Michiharu Nakano * and Yuichi Kanaoka Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060 (Japan) (Received 13 January 1987)

Key words: Acyl enzyme; Active site; Topography; Enzyme kinetics; Inverse substrate; Reporter group; Fluorescent; Trypsin

The preparation of fluorescence labeled acyl enzymes (Streptomyces griseus trypsin) was suceesdully carried out using specific trypsin substrates, 'inverse substrates'. The topographical analysis of the structures of the area around the active site was carried out by measuring the fltmrescence spectra of the acyl enzyme prepmv,tions and these results were compared with those of bovine trypsin. It was found that the ~ d y of the active site vicinity at pH 5 was similar to that of bovine trypsin, whereas considerable differences were noticed at lower pH as a result of pH-induced transformation. Conformational changes of the active site induced by the interaction with the specific iigand were analyzed from the fluorescence spectra. In these responses the two enzymes were quite distinguishable. The two enzymes active sites were also difh~nt in the energy transfer experiments. The spatial arrangements of the catalytic residues relative to the intrinsic tryptophan residues were suggested to be substantially different for the two enzymes.

Introduction Our early work [1] showed t h a t acyl derivatives of p-amidinophenol behave as specific substrates of trypsin, in spite of the fact that the site-specific cationic group for the enzyme is included in the leaving portion. Accordingly, the site-specific group is liberated during the enzymatic acylation stage to produce an acyl enzyme composed of nonspecific residue. A new term, 'inverse substrates', was proposed for these esters based on their structural characteristics. The design of 'inverse substrates' was extended to those carrying a * Present address: Hokkaldo Institute of Public Health, Sapporo, Japan. Abbreviation: Dns, 1,5-dimethylaminonaphthalenesulfonyl. Correspondence: K. Tanizawa, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan.

variety of reporter groups in the acyl part as a tool for the structural analysis of the enzyme, In a previous report [2], the preparation of acyl bovine trypsins in which a fluorescent group is designed to be attached to the enzyme catalytic residue through spacer groups of various chain lengths was successfully carried out using 'inverse substrates'. Topographical analysis of the structure of area around the active site was carried out by means of these fluorescence-labeled acyl bovine trypsin. Many enzymes are known to exhibit trypsin-like specificity, i.e., lysyl and arginyl bonds of substrate peptides are specifically hydrolyzed by these enzymes. Their active-site structures are assumed to be very similar, since the amino-acid sequences of the respective active sites are almost identical for these trypsin-like enzymes [3]. It is important to know the spatial features of the active sites and

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

293 to try to find some differences in their fine structures to enable the design of specific compounds which are able to discriminate one enzyme from others of similar specificity. We have therefore extended our approach to Streptomyces griseus trypsin, which is very close to bovine trypsin in structure and in catalytic function [4,5].

Preparation of acyl enzymes Preparation of acyl enzymes was carried out following the procedure previously reported [1].

Determination of remaining activity of acyl trypsin

Experimental procedure

Assay was carried out using N~-carbobenzyloxylysine p-nitrophenyl ester as substrate in 0.1 M citrate at pH 3.0 following the method of Bender et al. [17].

Materials

Spectrophotometric determination of number of acyl residue per tool enzyme

Bovine trypsin was obtained from Worthington Biochemical Corp. (twice crystallized, salt-free: TRL). S. griseus trypsin was prepared by purification of pronase P (Kaken Chemical Corp). Both enzymes were further purified through ST-Sepharose following an earlier procedure [14]. Titrated normality of the enzyme preparation with pnitrophenyl p'-guanidinobenzoate [15] was determined to be 92 and 90% on an optical absorbance basis for bovine and S. griseus trypsins, respectively. N~-Carbobenzyloxy-L-lysine pnitrophenyl ester hydrochloride was purchased from Aldrich Chemical Corp. Chemicals for synthetic work were obtained from Tokyo Kasei Industry, Tokyo. Buffers and solvents were of the best grade available for commercial sources. Synthesis of l a - e was carried out following the procedure reported previously [2]. Alkylguanidine sulfates were synthesized from alkylamine and S-methylisothiourea following the method of Phillips [16].

The following experimentally determined values were used for the determination: dansyl residue, E329n m 4730, e280n m 1680.

Measurement of fluorescence spectra Corrected fluorescence spectra were recorded on a Hitachi spectrofluorometer 650-60 using Rhodamine B as a standard. Measurements were carried out at 25 °C at the enzyme concentrations of 0.2 mg/ml. For the energy transfer analysis in which the shorter excitation wavelength (295 nm) was used, the enzyme concentration was lowered as much as 0.03 mg/ml. The optical absorbance at the excitation wavelength was less than 0.1 to ensure linearity in the fluorescence response. The following buffers were used for the pH-dependence experiments: 0.05 M acetate-HCl (pH 2-3); 0.05 M citric acid-Na2HPO 4 (pH 3-6).

Results Synthesis of fluorescent inverse substrates

Determination of kinetic parameters Bovine and S. griseus trypsin-catalyzed hydrolyses of l a - e was analyzed using a Union Giken Corp. stopped-flow spectrophotometer RA-401 and Hitachi spectrophotometer 200-10 equipped with thermostatically controlled water jacket. The reaction was carried out in 0.05 M Tris 0.02 M CaC12 1% dimethylformamide at 25°C. The enzyme concentration was 2.3 /~M and substrate concentrations were 41-155/~M. Kinetic parameters were determined by use of Lineweaver-Burk plots of the experimentally determined apparent rates following a procedure reported earlier [1]. Determination of activation parameters were carried out following an earlier procedure [7].

Synthesis of p-amidinophenyl esters in which the dimethylaminonaphthalenesulfonyl (dansyl) group was linked to a methylene chain of various lengths (la-d) was carried out by coupling the chlorocarbonate of dansylaminoalcohol with Nblocked p-amidinophenol and subsequent removal of the blocking group as previously reported [2].

Kinetic parameters for S. griseus trypsin-catalyzed reaction of la-d Compounds Ia-d were found to be susceptible to the catalysis by S. griseus trypsin in a specific manner. The reaction proceeded through the formation of a stable acyl enzyme intermediate. The

294 TABLE I

• z .,C-.~(, H2N

)>-O-C-O(CH2)nNH-SO

CHARACTERISTICS OF ACYL TRYPSINS

N(CH3) 2 I

a:

n=3

b: n : 4 C: n = 5

The data presented are mean values of three experimental series. S.E. < 4%. Acyl trypsin derived from

Remaining activity " (%)

la lb ic Id

1 1 3 3

d: n=6 Fig. 1. Design of specific acylating reagents (inverse substrates) labeled with fluorescence group.

rapid acylation process was analyzed with a stopped flow spectrophotometer by monitoring the liberation of p-amidinophenol at pH 8.0 and 25 ° C. Dissociation constants of enzyme-substrate complex, K s, and acylation rate constants, k E, were determined from double-reciprocal plots of the presteady-state acylation rates vs. substrate concentrations. Deacylation rate constants, k 3, were determined from the steady-state catalytic rates. The values for lb and le, for example, .are: K s, 4.0 pM and 1.8 pM; k2, 40 s - 1 and 20 s-I; k3, 2 . 2 × 1 0 -4 s -l and 8 . 3 x 1 0 -4 s -1, k 2 / k 3, 1.83 × 10 5 and 2.40 × 10 5, respectively. The K s values in the order of 10 -6 M exhibit strong binding affinity. Moreover, sufficiently large k 2 / k 3 ratios reflect predominant production of the acyl e0zyme intermediate. The kE//k 3 ratios for S. griseus trypsin were 10a-times larger than those previously reported for bovine trypsin [2]. This suggests that specific production of acyl enzyme intermediate is greater for S. griseus than for the bovine enzyme.

Preparation of acyl trypsins S. griseus trypsin was incubated with 15-20 molar excess of the ester for 2 min at pH 8.0 and 25 ° C. After the pH was adjusted to 2.0 by the addition of 1 M HCI, the reaction mixture was desalted by gel-filtration and lyophilized. The preparation was completely inactive as a result of acylation at the active site. The enzymatic activity was recovered completely after incubation for 2.5 h at pH 8.0 as a result of spontaneous deacylation. The observations were identical with those for bovine trypsin. The results are summarized in Table I together with the stoichiometry of the acylation determined spectrophotometrically.

Activity recovered after deacylation a,b

Acyl group introduced (mol/mol enzyme)

(%) 91 106 81 78

0.86 0.94 0.96 0.95

" Catalytic activity was analyzed using Na-carbobenzyloxy-Llysine p-nitrophenyl ester as a substrate at pH 3.0, 25 o C. b Deacylation was carried out at pH 8.0 for 2.5 h.

Structural analysis of the area around the S. griseus trypsin active site by means of fluorescent reporter groups - comparison with bovine trypsin Polarity of microenvironment. Excitation and emission maxima in the fluorescence spectra of the acyl trypsins in which the dansyl group is attached to the catalytic residue through spacer groups of various chain lengths were measured. At pH 5.0, emission maxima for acyl enzymes from Ia-c were not very different from each other (552-557 nm), but that from Ill was observed at the shorter wavelength (549 nm). The difference in the emission maxima of dansyl group could arise from a difference in the microenvironments in which the fluorophores reside. The polarity of the area around the active site was estimated using a Z-value scale [6] as Z = 90 for Ia-Ie (n = 3-5) and Z = 88 for Id (n =6), respectively. These values are exactly the same as those reported previously from bovine trypsin [2]. This reflects similarity of the active site structures at pH 5.0 of bovine and S. griseus trypsins with respect to the polarity. pH-Induced conformational change. The effect of pH on the emission maxima was investigated for the acyl trypsin (Fig. 2). For the case of acyl S. griseus trypsins derived from le and ld, an inflection of the curve was noticed at pH 3-4, whereas the emission maxima of the preparations from la and lb were almost pH-independent, The emission maximum of the acyl group itself is pH-indepen-

295 !

b

(3 •; 19.5 E I,J,

Ii

"-" 19

E 3

E

°--

~ 1a.5 E E

.9 18 ffl I/1 o_

E I

I

I

I

i

I

i

i

i

i

2

3

4

5

6

2

3

4

5

6

pH

pH

Fig. 2. pH-dependencyof emissionmaximaof acyltrypsins.(a) Acylbovinetrypsin;(b) acyl S. griseus trypsinfromIa (I), ib (O), lc (A) and Id (×).

dent [2]. Therefore, the observed pH-dependency could be due to the conformational changes of the enzyme active site regions caused by compounds Ic and Id. We propose that the region very close to the active serine residue is not flexible under changing pH, as shown by the spectra from compounds Ia and lb. These results are quite different from those of bovine trypsin, in which the conformational change reflected in the polarity change is more or less involved in all acyl enzymes derived from Ia-ld. In Fig. 2, results from bovine trypsin are also shown for comparison.

Effect of alkylguanidium ions on the deacylation rates and fluorescence spectra of acyl trypsins Acyl trypsins derived from 'inverse substrates' are distinct from those from normal substrates because the amidinophenyl moieties in the substrates are cleaved as a leaving group at the acylation stage the resultant acyl enzymes lack a cationic group to interact with the enzyme-binding site. Thus, the addition of an alkylamidinium or alkylguanidium ion to such acyl enzymes results in the formation of acyl enzyme-positive ligand complexes. The observation of rate acceleration at the deacylation step by the addition of cationic molecules was determined to be a complementary effect between the specific ligand and the intro-

duced nonspecific acyl group. Within the active site, both groups may simulate the deacylation of a cationic acyl enzyme, probably through a conformational change at the active site [7]. Addition of alkylguanidine derivatives also caused rate acceleration in the deacylation step of the fluorescent acyl enzyme. Parameters for the activation process, K i' and k ~ / k 3, which are denoted in eq. 1, were determined following the earlier procedure [7]. The values for bovine and S. griseus trypsins are listed in Tables II and III, respectively. Addition of the ligand was observed to cause a simultaneous spectral shift. Ks

. k2

k3

E+S~ES~E-A~E+ P

L

(1)

E-A-L

Addition of methyl-, ethyl-, n-propyl- and nbutylguanidinium ions resulted a shift of the emission maxima of acyl bovine trypsins derived from l a - d to longer wavelengths. The shift increased stoichiometrically as the concentration of the

296 TABLE II ACTIVATION PARAMETERS FOR BOVINE TRYPSIN CATALYZED HYDROLYSIS AT pH 8.0, 25 o C The data are mean values of three experimental series. S.E. < 8%. Ligand

Substrate:

Methylguanidine Ethylguanidine n-Propylguanidine n-Butylguanidine

la

lb

Ic

Id

K i' (raM)

k;/k 3

K~ (mM)

k~/k 3

K" (mM)

k~/k 3

g i' (mM)

k;/k 3

2.9 6.4 19 18

6.9 6.8 7.9 7.1

6.1 2.2 20 16

4.3 5.8 4.6 2.7

18 3.6 31 47

11 9.0 8.0 5,5

25 11 59 62

8.5 8.0 5.8 3,4

TABLE III ACTIVATION PARAMETERS FOR S. GRISEUS TRYPSIN-CATALYZED HYDROLYSIS AT pH 8.0, 25 o C The data are mean values of three experimental series. S.E. < 9%. Ligand

Substrate:

Methylguanidine Ethylguanidine n-Propylguanidine n-Butylguanidine

la

lb

le

ld

K i' (raM)

k;/k 3

g i' (mM)

k~/k 3

Ki' (mM)

k~/k 3

K.~ (raM)

k~/k a

1.8 0.30 0.21 2.0

54 159 36 12

19 7.0 2.6 62

57 83 45 28

91 7.9 3.1 15

7.1 10 3.0 2.4

15 12 69 58

11 9.9 3.8 2.0

ligand increased, although its extent, A?~, was not large (about 7nm). The half-saturation concentrations of the ligand, [L]~, for the spectral shift together with A h were determined (Table IV). Both parameters, K i' and [L]½, are in good agreement in each case as far as bovine trypsin is

concerned. Therefore, we deduced that the conformational change induced by the ligand interaction takes place at the broad region of the active site uniformally in the case of bovine trypsin. In the case of S. griseus trypsin, the conformational change accompanied with the complexation with

TABLE IV EFFECT OF ALKYLGUANIDINE ON THE EMISSION MAXIMA OF ACYL ENZYME AT pH 8.0, 25 o C

S. griseus

Ligand

Acyl trypsin from:

la

Ib

Methylguanidine

[L]½, " (raM)

1.5

5.0

8.0

9.0

U.D. b

U.D.

6.0

A h (nm)

3

4

4

3

0

0

3

2

[L]~ (raM) zlh (nm)

0.4 3

1.5 5

2.5 4

9.0 7

U.D. 0

U.D. 0

U.D. 1

U,D. 0

15 6

5

U.D. 0

U.D. 0

4.0 2

U.D. 0

30 2

5

U.D. 0

U.D. 0

Ethylguanidine

n-Propylguartidine

n-Butylguanidine

[L]_~ (raM)

Bovine la

AX (nm)

15 2

[L]_~ (raM) AX (nm)

25 3

Ib

9.0 5 12 5

le

Id

lc

~ 2

Id

15

80 2

" Ligand concentration which affords the spectral shift to the extent of Ah/2. Values are means of three experimental series (S.E. < 9:), b Undeterminable.

297

the ligand is smaller and the appearance of the change depends on the chain length of the acyl group. The acyl enzymes from la and lb did not show a shift of the emission maximum. The acyl enzymes from Ie and Id caused a slight shift. The values of [L]~ in these cases are ambiguous. It was noticed, moreover, that the addition of alkylguanidine derivatives at a concentration range of 10-5-10 -3 M caused a gradual non-stoichiometric blue shift of the emission maxima only in the cases of acyl S. griseus trypsin from Ia and lb. At the present stage, no reasonable explanation can be presented for this non-stoichiometric effect.

Energy transfer between tryptophan and dansyl residues Fluorescence spectra of trypsin itself exhibit the emission maxima at 300 nm due to the intrinsic tryptophan residues when excited at 295 nm. Since an excitation maximum of the extrinsic dansyl group is observed at 330 nm, the excitation of tryptophan at 295 nm will eventually excite emission of dansyl group at 550 nm. Thus, the fluorescence intensity of tryptophan residues at 330 nm is reduced for acyl trypsin ( F ) compared to that for native trypsin (F0). This extent is related to the energy transfer efficiency (T) following Eqn. 2 [8]. T= I - ( F/Fo)

(2)

Values of energy transfer efficiency for S. griseus trypsin were determined and these were compared with the previous data for bovine trypsin (Table V). It is known that the efficiency of energy transfer is sensitive to the spatial relationship between TABLE V C O M P A R I S O N OF E N E R G Y T R A N S F E R EFFICIENCIES F R O M T R Y P T O P H A N R E S I D U E TO E X T R I N S I C DANSYL G R O U P BETWEEN STREPTOMYCES GRISEUS TRYPSIN A N D BOVINE TRYPSIN Acyl trypsin derived from:

Energy transfer efficiency (5~)

S. griseus

bovine

la

73

36

lb Ic Id

73 65 60

38 39 48

donor and acceptor molecules. Sharp contrast between these two trypsins was observed. The efficiency for S. griseus trypsin was much higher than that for bovine trypsin. A dansyl group connected with the longest methylene chain (Id) exhibiting highest efficiency may suggest the closest proximity to the tryptophan residues in the case of bovine trypsin. In contrast, ld resulted in the lowest efficiency in the case of S. griseus trypsin. Discussion

The similarity of S. griseus and bovine trypsins in their structures as well as in their catalytic characteristics is well recognized, and the aminoacid sequence identity was approx. 33% [5]. An X-ray crystallographic study revealed that the global structures of both enzymes are very similar, in spite of the fact that S. griseus trypsin has three disulfide bridges whereas bovine trypsin has five [9]. In the comparative model studies for both enzymes, it was analyzed that the amino-acid sequence was divided into homologous and non-homologous parts in each model and that individual homologous segments have very similar conformation but differ slightly in their orientation and position relative to other homologous segments [9]. From analysis of the molecular model of bovine trypsin constructed from the X-ray diffraction data [10], the enzyme active site cavity was shown to be composed of three peptide segments [3]: Ala55-Tyr-59, Asp-189-Gly-197 and Gly-211-Ala220 (Fig. 3). Three segments in S. griseus trypsin equivalent to those in bovine trypsin were determined. Within these segments for both enzymes, the amino-acid sequence identity is 21 out of 24 residues (88% identity): Tyr-59, Ser-190 and Ser-217 in bovine trypsin are replaced by Val, Thr and Tyr in S. griseus trypsin, respectively. Our observation that the polarity of the area around the active site responded differently to pH changes could be due to such a minor difference in the amino acid sequence. It is also suggested from the molecular model that the decapeptide, Tyr-94-Ile103, is a candidate for the dansyl-interacting site of bovine trypsin. The peptide is shown in Fig. 3. This segment is located at the outer limb of the binding cavity in the region 10-15 ,~ different from the catalytic serine residue. This distance

298

tyS-Sb~'

s,~-l@2

Thr-98

Tyr-94 I~-

(Lys)

corresponds to that between the dansyl and carbonyl of the acyl groups for Ia-d in their fully extended conformations. The sequence identity of the decapeptides within two enzymes is as low as 50%. Both S. griseus and bovine trypsins contain four tryptophan residues. Two of them in each enzyme are at the homologous segments; Tip-141 is at the back-side of the binding cavity in a completely buried state and Trp-215 is at the binding cavity, which is the most favorable position for energy transfer, as shown in Fig. 3. It may be assumed, therefore, that the difference in energy transfer efficiency between these two enzymes is not due to these tryptophan residues, assuming that the orientations of the indole rings relative to the dansyl group are alike for both enzymes. (Close similarity of the orientation of the indole rings is estimated from the X-ray diffraction data; both the a-carbon atoms of Trp-215 and Trp-141 for S. griseus trypsin were superim-

Fig. 3. Drawing of peptide segments constructing the binding cavity of bovine trypsin on the basis of molecular models (Nicholson; LABQUIP). Open circles represent position of a-carbons. Side-chains of several aminoacid residues are also shown, The active-site structure of S. gr/seus trypsin was estimated on the basis of the identity of amino-acid sequences for both trypsins. Amino acids in parentheses are those of S. gr/seus trypsin not equivalent to those in bovine trypsin. The numbering system is that of chymotrypsinogen A.

posed on the corresponding atoms for bovine trypsin within 1 A distance [19].) The other two residues are variable; for S. griseus trypsin they are at the positions 103 and 207; for bovine trypsin they are at 51 and 237. The region including 51 for bovine trypsin and the region including 207 for S. griseus trypsin are at opposite sides of the cavity of the respective molecules and they are assumed to be only slightly involved in the energy transfer. As shown in Fig. 3, the residue at 103 is rather close to the cavity, although it is buried. The enhanced energy transfer efficiency for S. griseus trypsin, therefore will be explained by the additional contribution of Trp-103. The dansyl group is closer to Trp-103 when the acyl group with a shorter spacer group is present. The alternative tryptophan residue for bovine t.rypsin is at position 237, which locates about 5 A apart from the cavity than 103. A slight improvement in the proximity of the dansyl group to the buried Trp-237 is expected in the case of

299 the longest acyl group. The energy transfer efficiency depends on the spatial relationship of d o n o r and acceptor [11]. However, we currently need to use an a p p r o x i m a t i o n that the array of b o t h groups are averages of the r a n d o m orientation. In fact, estimation of possible orientation of dansyl group in the acyl trypsin model is not feasible, since dansyl moiety has shown to have a lot of rotational freedom. For the enzymes of trypsin-like specificity, 'inverse substrates' have provided a general m e t h o d for the efficient production of acyl enzyme without recourse to the structure of acyl group [1]. In this report, the m e t h o d has been shown to be applicable to the analysis of topographical structure of the area a r o u n d the trypsin active site. U n f o r t u n a t e l y we have not succeeded in designing 'inverse' type substrates for the enzymes other than trypsin-like specificity, except the case of butyrylcholinesterase [12]. An interesting finding, mirror image catalysis, for glyoxalase has been reported by Kozarich and Chaff [13]. It is reasonably assumed that enzyme could not always respond rationally towards a variety of synthetic substrates and inhibitors. The finding b y Kozarich and Chaff as well as o u r observation with 'inverse substrates' m a y be considered to be due to such an imperfection of enzymes.

Acknowledgements This work was supported in part by a Grant-inAid for Special Project Research from the Ministry of Education, Science and Culture, Japan, and grants f r o m the M o c h i d a Memorial F o u n d a t i o n for Medicinal and Pharmaceutical Research, the

F o u n d a t i o n of Research for Medicinal Resources and the F u g a k u Trust for Medicinal Research.

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