[52] Labeling of Δ5-3-Ketosteroid isomerase by photoexcited steroid ketones

[52]

AS-3-KETOSTEROID ISOMERASE

469

[521 Labeling of As-3-Ketosteroid Isomerase by Photoexcited Steroid Ketones B y WILLIAM F. BENISEK

The A'5-3-ketosteroid isomerase of Pseudomonas testosteroni catalyzes the allylic isomerization of A'~- and Al°-3-ketosteroids to the ±~ isomers. The enzyme has been extensively studied structurally and mechanistically for many years. Much of what is known about the enzyme from these studies has been comprehensively reviewed by T a l a l a y and Benson. ~ Several attempts to identify amino acid residues comprising the substrate binding site of the isornerase have been made using affinity labeling techniques. These include the use of 6B-bromotestosterone acetate as an active site-directed alkylating agent, 2 and the use 3 of 5,10-secoestr-5-yne3,10,17-trione and 5,10-seco-19-norpregn-5-yne-3,10,20-trione as kent inhibitors. 4 Pursuing a different approach, M a r t y r and Benisek '~,6 investigated the use of ±4-3-keto steroids, products of the isomerase reaction, as photoexcitable affinity reagents. These workers found that several members of this class of steroids stimulated an ultraviolet light-dependent photoinactivation of the enzyme. The loss of enzymic activity could be correlated with destruction of a single residue of aspartic acid or asparagine. Theory In their electronic ground state, saturated and unsaturated ketones possess limited chemical reactivity toward protein functional groups, reactions being limited to the addition of nucleophiles across the carbonoxygen double bond (as in Schiff's-base formation) and across a conjugated carbon-carbon double bond. The reactivity of the keto group can be enormously increased by the absorption of light. The n---> ,~*: absorption band of ketones is centered in the range of 280-320 nm, the exact position depending upon the structure of the ketone and the solvent. Absorption of a photon in this wavelength range and at somewhat longer 1p. Talalay and A. M. Benson, "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 6, p. 591. Academic Press, New York, 1972. -~K. G. Btiki, C. H. Robinson, and P. Talalay, Biochim. Biophys. Acta 24'2, 268 (1971). 3F. H. Batzold and C. H. Robinson, J. Am. Chem. Soc. 97, 2576 (1975). 4R. R. Raado, Scie~,ce 185, 320 (1974). See also this volume [3] and [12]. 5 R. J. Martyr and W. F. Benisek, Biochemistry 12, 2172 (1973). R. J. Martyr and W. F. Benisek, J. Biol. Chem. 250, t218 (1975).

470

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[52]

wavelengths results in excitation of one of the nonbonding electrons of the oxygen to the antibonding ,~*' orbital. The initially produced excited state is a singlet. It is a very short-lived species (lifetime of about 10-9 scc), which is efficiently transformed to the triplet state. The triplet state is much more stable (lifetime -- 10-3 to 10-2 sec at 77°K), and it is from this state that much of the photochemistry of ketones proceedsJ Since .the once paired nonbonding electrons of the ground state are in different molecular orbitals in the triplet excited state, this state exhibits a "diradical-like" character in its chemical properties. The unpaired electron on the oxygen imparts an alkoxy radical character to the ketone oxygen. Thus the first step in many of the photochemical reactions of ketones involves hydrogen atom abstraction from suitable donors by the oxygen atom generating a pair of free radicals. The radical pair can combine with each other, undergo additional hydrogen atom transfers, or other molecular rearrangements to yield the final products. If the hydrogen atom initially abstracted is part of a protein which binds the ketone, then photoaffinity labeling of the protein by the excited ketone is, in principle, possible. A very large body of literature exists on the photochemical reactions of ketonesJ -1° Only a brief indication of reactions that might be of importance in affinity labeling of proteins by A4-3-ketosteroids will be presented here. Addition o] Nucleophiles 11-13 OCH3

~ " 0 0

~ ~

0 ~ " 0

"" ~

0

o

II

H t-BuOH tBu

7 See, for example, J. A. Barltrop and J. D. Coyle, "Excited States in Organic Chemistry." Wiley, New York, 1975. s R. O. Kan, "Organic Photochemistry." McGraw-Hill, New York, 1966. o O. L. Chapman and D. S. Weiss, in "Organic Photochemistry" (0. L. Chapman, ed.), Vol. 3, p. 197. Dekker, New York, 1973.

[52]

•5-3-KETOSTEROID

ISOMERASE

471

A d d i t i o n o / H y d r o c a r b o n C - - I I B o n d s '4-17 0Ac

0Ac

> 0

0

Ctt2

(5

@_1

0

h,~ CH3~

C ~

CH3

12113

C - - OH

+

I Ctt3

0

0

II

II

0

h~

n ~-C-H

+

N-acetylglyclne

methyl

ester

~

0

I..1 0

II

III

CH3-C-NH-C-C-OCH3

I ~-c-H

I OH

1oN. J. Turro, J. C. Dalton, K. Dawes, G. Farrington, R. Hautala, D. Morton, M. Niemczyk, and N. Schore, Acc. Chem. Res. 5, 92 (1972). ~1T. Matsuura and K, Ogura, J. Am. Chem. Soc. 88, 2602 (1966). 1=T. Matsuura and K. 0gura, Bull. Chem. Soc. Jpn. 40, 945 (1967). ~ W. G. Dauben, G. W. Shaffer, and N. D. Vietmeyer, J'. Org. Chem. 33, 4060 (1968). ~' D. Bellus, D. R. Kearns, and K. Schaffner, Helv. Chim. Acla 52, 971 (1969). is N. C. Yang and D. D. H. Yang, J. Am. Chem. Soc. ~}, 2913 (1958). ~ S. Wolff, W. L. Schreiber, A. B. Smith III, and W. C. Agosta, ]. Am. Chem. $oc. 94, 7797 (1972). ~7R. E. Galardy, L. C. Craig, and M. P. Printz, Nature (London), New Biol. 242, 127 (1973).

472

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[52]

Photochemical Oxidation-Reduction 16,18

0~

0 I~

J

o

I J It

o

o

o

hv

Based on the foregoing reactions, some useful observations pertaining to the use of A4-3-ketosteroids as affinity reagents can be made. First, the chemical specificity of the photoexcited keto group is very broad since both nucleophiles and C - - H groups are potential reactants. This would suggest that an enzyme's binding site need not contain reactive nucleophiles in order to be labeled. Second, reactions of the photoexcited steroid with a protein may or m a y not result in covalent attachment of the steroid to the protein. Third, the possibility of occurrence of nonspecific side reactions is a very real one, particularly in the case of a species that is as reactive as a ketone excited state. Equipment

Light Source and Filters Since the n --> ~r* band of ketones is in the 280-320 nm range, a light source that emits radiation in this region is necessary. For many purposes 1~R. Breslow, S. Baldwin, T. Flechtner, P. Kalicky, S. Liu, and W. Washburn, J. Am. Chem. Soc. 95, 3251 (1973).

[52]

A5-3-KETOSTEROID ISOMERASE

473

we have found a medium-pressure mercury are lamp to be adequate. A suitable lamp is manufactured by Hanovia Lamp Division, Canrad Precision Industries, Inc., Newark, New Jersey TM (Model 679A, rated at 450 watts). According to the manufacturer it produces an are 41/./z inches long. The emission spectrum of the lamp is not continuous, but consists of a large number of discrete lines extending from the infrared through the ultraviolet. The most intense lines are located at 1014 nm, 578 nm, 546 nm, 436 nm, 404.5 nm, 366 ran, 334 nm, 313 nm, 302.5 nm, 297 nm, 280 nm, 265 nm, 254 nm, and 222 nm. The envelope of the lamp is made of quartz, which will pass all these wavelengths. It is therefore necessary to block those wavelengths likely to excite protein ehromophores, a process known to result in the inactivation of many enzymes, z° On the other hand, radiation effective in exciting the A~-3-keto group [,k...... ( n - ~ ~'~) ~ 310 nm with a long wavelength tail extending to about 360 nm] must be transmitted to the sample. One useful filter that possesses suitable absorption characteristics is Pyrex glass (Coming 7740), which absorbs essentially all wavelengths below 280 nm and exhibits partial absorption of radiation between 280 and 350 nm. According to Calvert and Pitts, :~ 4 mm of Pyrex glass transmits 10% of incident 310 nm radiation, 30% at 319 nm, and 50% at a30 nm. The Hanovia lamp is cooled during use by means of a glass immersion well (dewar type) in which it operatesY,) This is a double-walled Pyrex or quartz dewar between the walls of which cooling water is circulated. When, as is usual, a Pyrex dewar is employed, 4 mm of Pyrex are traversed by the radiation. Additional filtration can be achieved by the insertion of glass filter sleeves TM between the lamp and the innermost wall of the immersion well. Solution filters can also be employed, and several suitable for use in the ultraviolet are described by Calvert and Pitts. 2~ Katzenellenbogen et al. 2~ used a saturated solution of cupric sulfate to screen out wavelengths shorter than 315 nm. R e a c t i o n Vessels

Pyrex nuclear magnetic resonance (NMR) sample tubes (0.4 mm wall thickness) are suitable for containing the sample to be irradiated. These can be purchased from Kontes Glass Co. in several different grades. The tubes can be stoppered with rubber septa during experiments requiring anaerobic conditions. 19Lamps, immersion wells, and filter sleeves are available from Ace Glass Company, Vineland, New Jersey 08360. ~ R. Setlow and B. Doyle, Biochem. Biophys. Acta 24, 27 (1957). :1 j. G. Calvert and J. N. Pitts, Jr., "Photochemistry." Wiley, New York, 1966. ~'~J. A. Katzenellenbogen, I-I. J. Johnson, Jr., K. E. Carlson, and H. N. Meyers, Biochemistry 13, 2986 (1974).

474

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[52]

SCW

TO

POWERS

SC~

SIDE VIEW

L

W

H

TOP VIEW

FIG. 1. Apparatus used for photoaffinity labeling. Shown are schematic scale drawings of side and top views of the photochemical reactor in current use. The

[52]

AS-3-KETOSTEROID ISOMERASE

475

Photolysis Apparatus Early experiments ~ employed an apparatus in which the lamp and sample were contained in a light-tight wooden cabinet. The distance between the center of the mercury arc and the center of the sample tubes was 8.5-9.0 cm. Up to three tubes could be irradiated at the same time. The lamp was surrounded by a quartz cooling jacket, and the sample tubes were mounted in a Pyrex dewar flask that had a unsilvered window facing the lamp. Sample temperature was maintained by blowing coot dry nitrogen through the Pyrex dewar. This apparatus performed satisfactorily but suffered from several disadvantages. In order to protect personnel from exposure to dangerous radiation, the lamp had to be extinguished before the cabinet could be opened in order to withdraw aliquots from the samples. Only three samples could be irradiated simultaneously, and sample temperature control was rather poor ( ± 5 ° C ) . This apparatus was superseded 6 by a "merry-go-round" type of device that does not have these disadvantages (Fig. 1). The lamp, L, is mounted vertically at the center and is surrounded by a filter sleeve, F, which, in turn, is surrounded by the Pyrex immersion well, W. Tap water flows between the walls of the immersion well in order to dissipate heat from the lamp. Surrounding the immersion well is a cylindrical "merry-goround" tube holder, H, drilled with 20 holes (4.5 cm radial distance) for the N M R tubes, T. The holder is mounted on nylon ball bearings so that it can rotate about a vertical axis passing through the lamp. The holder is driven at 6 rpm by a small electric motor (not shown) coupled to the rim of the holder by a rubber rimmed wheel. Rotation of the holder about the lamp serves to average out any angular inhomogeneity in the light. Thus all tubes receive the same dose rate averaged over time. The entire apparatus below the mounting plate, P, is surrounded by a stainless steel bath, B, which is filled with distilled water. This water is circulated to a thermostatically controlled water baths, Forma Model 2095, and serves to control the sample temperature. The lamp is energized by an Ace Glass Co. mercury vapor lamp power supply (Cat. No. 6515-60). General Procedures

Solutions to be irradiated are prepared in small test tubes from stock solutions of steroid (in ethanol), isomerase, and buffer and appropriate meaning of the identifying symbols is as follows: L, mercury arc lamp; F, filter sleeve; W, immersion well; H, sample tube holder; T, NMR sample tube; B, water bath; PT, photographic thermometer; P, mounting plate; SCW, sample tube cooling water (direction of flow indicated by arrows); LCW, lamp cooling water (direction of flow indicated by arrows).

476

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[52]

volumes (0.1-1 ml) transferred to N M R tubes. In the case of anaerobic samples the stock solutions are deoxygenated by bubbling nitrogen gas through them, and suitable aliquots are transferred directly to the N M R tubes under a nitrogen barrier. A nitrogen-purged glove box is convenient for this purpose. All tubes are securely stoppered with rubber septa. An identical set of tubes is prepared to serve as dark controls. The lamp cooling water and circulating bath water are turned on, followed by the lamp itself. After about 1 hr the lamp has reached a steady output, and the sample tubes are inserted into the holder and rotation begun. Zero time samples are withdrawn just prior to commencement of irradiation. Additional samples may be withdrawn at time intervals, care being taken to maintain the nitrogen atmosphere of the anaerobic samples.

Application to Affinity Labeling of As-3-Ketosteroid Isomerase

Kenetics of Inactivation Tubes containing purified 23 isomerase either alone or in the presence of various steroid competitive inhibitors are irradiated with 4 mm Pyrexfiltered light. At various times aliquots are withdrawn from the tubes, diluted with neutral 1% bovine serum albumin, and assayed 5,2~ for enzyme activity. The inactivation observed follows first-order kinetics. The first-order rate constants of inactivation are summarized in the table. K I N E T I C S OF PHOTOINACTlVATION a

K1 b

kapp

Addition

(pM)

(hr -1)

None Cyclohexenone Testosterone 19-Nortestosterone 19-Nortestosterone acetate

-> 5000 48 13 7

N 0 . 001 N 0 . 006 0. 023 0.06; 0.07 0.10

a Reaction mixtures contained 5 ~M isomerase, 3.3% ethanol, and 0.04 M sodium phosphate, p H 7.0, plus additions. The concentrations of the additions were cyclohexenone, 330 uM; testosterone, 23 ~M; 19-nortestosterone, 24 ~M; 19nortestosterone acetate, 21 ~M. Final volumes were 0.15 ml. Irradiations were performed using the "old" apparatus (see text). b K1 values were determined under standard assay conditions by the method of M. Dixon [Biochem. J. 55, 170 (1953)]. 23R. Jarabak, M. Colvin, S. H. Moolgavkar, and P. Talalay, this series, Vol. 15, p. 642 (1969).

[52]

A5-3-KETOSTEROID ISOMERASE

477

Three steroids that lack the A4-3-keto chromophore [3fl-hydroxy-5androstene-17fl-carboxylie acid, 3-methoxy-l,3,5(10)-estratrien-17fl-ol, and 3fl-hydroxy-5-pregnen-20-one] and are competitive inhibitors of the enzyme do not stimulate photoinactivation of the enzyme to a rate greater than that of a steroid-free control. The stimulation of photoinactivation required a steroid that bound at the catalytic site and possessed the ±~-3-keto chromophore. Additional results were interpreted 5 to indicate that the inactivation was A~-3-ketosteroid dependent and was probably active site-directed. Cyclohexeneone which possesses the same chromphore as ±4-3-ketosteroids but is not a competitive inhibitor of the enzyme, stimulated photoinactivation only to a small extent even when present at more than ten times the concentration of compounds that markedly stimulated photoinactivation. It was noted that the first-order rate constant for photoinactivation increases with increasing affinity of the enzyme for the particular steroid, a pattern to be expected if the photoinactivation is active site-directed. In addition, the rate of photoinactivation promoted by 19-nortestosterone acetate was decreased approximately 2-fold in the presence of 21 t,M 3fl-hydroxy-5-androstene-17fl-carboxylic acid, a competitive inhibitor that does not support photoinactivation of the enzyme. This protective effect further strengthens the conclusion that photoinactiration is a catalytic site-directed reaction. Partial Characterization of Photoinactivated Isomerase ~

Chemical changes accompanying the 19-nortestosterone acetate-promoted photoinactivation were monitored by amino acid analysis. It was observed that both aspartic acid and histidine decreased with increasing extent of inactivation. In two kinetic studies comparing the rates of activity, aspartic acid and histidine losses, it was observed that the firstorder rate constants for activity loss and loss of one residue of aspartate (asparagine) were nearly the same (k,~c~,.~ty= --0.54 hr ~, --0.55 hr~'; k~, = - - 0 . 5 6 hr -~, --0.52 hr -') whereas the rate of histidine loss was substantially slower (k~,~.~= - - 0 . 1 6 hr -~, --0.13 hr-~), suggesting that aspartate destruction was responsible for the inactivation of the enzyme. Certain controls were run which indicated that aspartate destruction is a steroid binding site-specific process. Thus irradiation of isomerase in the presence of cyclohexenone, 3fl-hydroxy-5-androstene-17fl-carboxylic acid, or both together did not result in aspartate destruction (or in histidine destruction). Also, irradiation of performic acid oxidized bovine pancreatic ribonuclease A, to which steroids presumably do not bind, in the presence of 19-nortestosterone acetate induced no destruction of

478

ENZrMES, ANTIBODIES, AND OTHER PROTEINS

[52]

aspartic acid. Attempts to more fully characterize the nature of the aspartate-destroying reaction have been made recently. No significant differences are seen between peptide maps of the soluble tryptic peptides of native and photochemically inactivated isomerase. From chymotryptic digests of the insoluble material remaining after tryptic digestion, a peptide whose amino acid composition corresponds to residues 31-4554 has been purified. 2~ A similar peptide has been purified by the same procedure from the inactivated enzyme. The peptide .from the photoinactivated enzyme contains 0.7 fewer residues of Asx and 0.7 more residues of alanine than the corresponding peptide from the native enzyme, suggesting that the aspartate destroying reaction includes a decarboxylation or decarboxamidation. Experiments to clarify the chemistry of the aspartate (asparagine) destroying reaction and its position in the polypeptide chain are in progress. Ef]ects o] Oxygen s

The presence of oxygen was found to markedly influence the character of the photochemical events occurring during photoinactivation promoted by 19-nortestosterone acetate. Oxygen inhibited both the loss of activity and the destruction of aspartate whereas it stimulated destruction of histidine. Under anaerobic conditions, no significant loss of histidine occurred whereas 0.98 of 12 moles of aspartate (per mole of enzyme monomer, M W 13,394) was destroyed and 85% of the activity was lost.

Related Applications Several reports describing photoa~nity labeling studies in which the photoactivation of ketones play a role have appeared recently. Glover et al. 2~ have found that partially active ~-chymotrypsin derivation containing phenacyl, 1-naphthacyl, and 2-naphthacyl moieties on the sulfur of methionine 192 (as sulfonium salts) undergo partial reactivation upon irradiation with A >300 nm using an apparatus similar to the one described here. They found that about half of the protein-associated phenacyl group could be detached from the protein by irradiation, yieldu This sequence is --- Ala Asp Asn Ala Thr Val Glu Asn Pro Val Gly Ser Glu Pro Arg ---. ~ T h e peptide was purified by paper chromatography (butanol/acetic acid/H~O 4:1:5 upper phase) followed by p H 6,5 electrophoresis at a right angle to the direction of chromatography, and then pI-I 1.9 electrophoresis. ~ G. I. Glover, P. S. Mariano, T. J. Wilkonson, R. A. Hildreth, and T. W. Lowe, Arch. Biochem. Biophys. 162, 73 (1974). See also this volume [71].

[53]

&NTI-DINITROPHENYL A.NTmODIES

479

ing fully active ~-chymotrypsin. They proposed that in addition to this reaction a concurrent photoprocess occurred yielding an inactive species. This process may involve an uncharacterized photochemical reaction between the excited keto group of the phenacyl moiety and a nearby amino acid side chain. Galardy et al. 17,27 reported that pentagastrin derivatives, 4-acetylbenzoyl pentagastrin and 4-benzoylbenzoyl pentagastrin, became covalently attached to bovine plasma albumin when irradiated with ultraviolet light. The photoattachment of the 4-acetylbenzoyl derivatives appeared to possess some specificity since oleate provided a substantial inhibition of the attachment process. Katzenellenbogen et al. 22 found that 6-oxoestradiol inhibited the estradiol binding activity of rat uterine cytosol during irradiation with )~ > 315 nm and that the inhibition reaction was strongly suppressed by inclusion of estradiol in the photolysis reaction mixture. ~7R. E. Galardy, L. C. Craig, J. D. Jamieson, and M. P. Printz, J. Biol. Chem. 249, 3510 (1974).

[53] A f f i n i t y L a b e l i n g o f A n t i b o d y C o m b i n i n g S i t e s as Illustrated by Anti-Dinitrophenyl Antibodies B y DAVID GIV0L and MEIR WILCHEX

The chemical structure of the combining site of antibodies was a central paradox in immunology: How could an apparently infinite range of combining specificities reside in what appeared to be a very monotonous group of proteins, closely related in their structure? Considered from this viewpoint, it is interesting that, relative to other proteins with combining sites, e.g., enzymes, antibodies are a late evolutionary acquisition, present only in vertebrates, and their synthesis is confined to only one type of cell: the lymphocyte. If we compare, on the one hand, the number of different combining sites that are being made by all cells of our body, and on the other, the number of different combining sites that lymphocytes can make in the form of antibodies, it is clear that the lymphocytes can produce a greater variety of proteins than all the other cells in toto. This is evident since the immune system can make more than one antibody against any specific foreign protein. It has therefore been a long-standing question whether the structure of the antibody site is based on principles that differ from those governing the structure of the active sites of other proteins. All immunoglobulins have the same basic multichain subunit, a symmetrical molecule composed of two heavy chains (H) of molecular weight