We Utilized a Similar Approach with DEP-Chymotryp- Two of The

PROBING THE TOPOGRAPHY OF THE ACTIVE SITE OF a-CHYMOTRYPSIN BY BERNARD F. ERLANGER DEPARTMENT OF MICROBIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY Communicated by Erwin Chargaff, May 16, 1967 This paper is a report of a study of the topography of the active center of a- chymotrypsin. In particular, it will deal with that portion involved in substrate binding and with its position in space relative to the nucleophilic serine residue that is believed to function in the catalytic mechanism.1 Since an enzyme must accommodate a substrate, the simplest approach to an understanding of the topography of the former might be a study of the conformation of the latter. However, this is not feasible with chymotrypsin. A typical substrate of chymotrypsin is acetyl L-phenylalanine ethyl ester.1 There is much evidence linking the susceptibility of this substrate to the aromatic ring of the amino acid (though aromaticity is not necessary since cyclic hexahydro derivatives are also substrates)2 and to the acylamido portion of the molecule. Benzoylamido may substitute for acetylamido and, in fact, yields a somewhat better substrate (higher keat)3 in the case of acyl L-tyrosine amide. However, since all of the above substrates are flexible molecules and therefore able to assume many conformations, there is no way of knowing their conformation when bound to the active center of a-chymotrypsin. In our early studies we chose to use an approach based upon the experiments of Wilson with acetylcholinesterase.4 Acetylcholinesterase and chymotrypsin belong to the class of enzymes called serine esterases.1 All members of this class contain an unusually reactive serine residue which acts as a nucleophile in the catalytic mechanism. They are inactivated by certain organophosphorus compounds by a reaction that can be understood within the context of the enzymatic mechanism of the serine esterases. Briefly, they participate in the acylation step of the mechanism but deacylate at an extremely slow rate unless nucleophiles considerably stronger than water are introduced, e.g., hydroxylamine, oximes, or hydroxamic acids. Wilson4 had shown that the specificity sites of diethylphosphoryl (DEP)-cholin- esterase were still available for binding of substrate-like molecules. It was possible for him to design reagents which were bound by the inactivated (phosphorylated) enzyme in such a way as to position their nucleophilic functional group one bond length away from the phosphorus atom, thus ideally situated for a nucleophilic displacement reaction. From a consideration of the structure of his best reactivators, he was able to map out some details of the topography of the active center of acetylcholinesterase. In our early experiments,' we utilized a similar approach with DEP-chymotrypsin. A large number of oximes and hydroxamic acids were tested as reactivators. Two of the compounds were found to be unusually reactive: N-phenylbenzohydroxamic acid and N-phenylnicotinohydroxamic acid. They were, respectively, 15 and 8 times faster reactivators than either benzohydroxamic acid or N-phenyl- 703 Downloaded by guest on September 26, 2021 704 BIOCHEMISTRY: B. F. ERLANGER PROC. N. A. S. acetohydroxamic acid. Hence, it was tentatively concluded that DEP-chymotrypsin had two sites capable of binding aromatic rings and that when it did so with N-phenylbenzohydroxamic acid and N-phenylnicotinohydroxamic acid, the nucleophilic hydroxamic acid group was favorably situated for a displacement of the phosphate group. Supporting evidence was obtained from experiments with DEP-trypsin.6 Since the specificity of trypsin is entirely different from that of chymotrypsin, N-phenylbenzohydroxamic acid would not be expected to be more effective than benzohydroxamic acid or aliphatic hydroxamic acids. It was, in fact, somewhat less active. Thus, our early experiments allowed us to conclude that the active center of a- chymotrypsin contained two ring structure-binding sites that could be utilized by the enzyme in its catalytic mechanism. However, their positions in space relative to each other and to the active serine residue could not be deduced because of the structural flexibility of the reactivator molecules. Later findings enabled us to define the topography more exactly. In 1963, it was found that diphenylcarbamyl chloride (DPCC) was a specific inactivator of chymotrypsin.7 Like the organophosphates, it inactivated the enzyme by a process that made use of the catalytic mechanism of the latter. Subsequent studies by Metzger and Wilson8 showed that methyl phenylcarbamyl chloride was also an inactivator of a-chymotrypsin but that it was about 150 times less active than DPCC, indicating again the presence on the enzyme of two ring- binding sites which could orient the carbonyl carbon properly with respect to the reactive serine. Construction of a space-filling model of DPCC revealed considerable steric interference with the freedom of rotation of the two aromatic rings, but still enough freedom remained to make an unambiguous mapping of the active center unfeasi- ble. Encouraged by these findings, we set about to design a completely constrained inactivator. Among the compounds synthesized was phenothiazine-N-carbonyl chloride (PCC),9 the structure of which is given in Figure 1, along with several other reagents specific for chymotrypsin. Its resemblance to DPCC is apparent; however, the additional sulfur bridge restricts the movement of the two aromatic rings completely. Even rotation around the N-C bond is restricted severely by neigh- boring hydrogens on the beuzene rings. Hence, fixing onie aromatic ring determines the 1)ositioIl in space of the susceptible carbonyl chloride bond. iPCC is an effective and specific inactivator of chymotrypsin. The second-order rate constant for its inactivation of a-chymotrypsin (105 liter mole-' sec-) is about one fourth that of DPCC. However, as shown by its reaction with OH-, it is inherently less reactive than DPCC by a factor of about 20. Correction for this lower reactivity in nucleophilic reactions leads to the conclusion that lPCC is even a more specific reagent for a-chymotrypsin than is DPCC. Another interesting facet of the reaction of PCC with a-chymotrypsin is that spontaneous reactivation occurs at a slow but measurable rate (k4 = 25.3 X 10-4 min' at 370, pH 7.55). PCC, therefore, is more like a susceptible substrate. This would imply that less distor- tion of the active center occurs as a result of acylation of the enzyme by PCC than by DPCC. Downloaded by guest on September 26, 2021 VOL. 58, 1967 BIOCHEMISTRY: B. F. ERLANGER7705 Cl Cl C=O C=O Diphenylcorbamyl Phenothiazine -N- Corbonyl Chloride Chloride 0 A tteC5OCHI H C-OCH3 Il"?H NH 0 0 iIi D-i-Keto-3-Corbomethoxy- o 0- l,2,3,4,-Tetrchydroieoquinoline IfOH Benzoyl L-Phenylalanine C1. .,HMethyl Ester N -Phenylbenzohydroxomic Acid FIG. 1.-Chemrical structures of specific substrates, inactivators, and reactivators of chymotrypsin. If we now construct models of all of the reagents shown in Figure 1, it becomes possible to map the topography of the active center of chymotrypsin. Among the reagents included is the substrate discovered by Hein et al.,'0 D-1-keto-3- carbomethoxy-1,2,3,4-tetrahydroisoquinoline (D-CDIC). D-CDIC can be looked upon as a D-phenylalanine analogue of benzoyl L-phenylalanine methyl ester with one aromatic ring serving a dual role as the acylamido group and as the benzene ring of phenylalanine. Surprisingly, the L-enantiomorph was a poor substrate. Wilson and ErlangerII provided an explanation for this unusual situation by postulat- ing that the aromatic ring was bound as if it were the benzoylamido group of benzoyl L-phenylalanine ethyl ester. This orientation will be utilized in the present discussion. An important aspect of this substrate is its highly constrained structure and hence its potential utility as a mapping tool. A model of benzoyl L-phenylalanine methyl ester was constructed in order to find a conformation that could be assumed by all of the constrained reagents. Only one could be found; it is shown in Figure 2. The molecule on the upper left is phenothiazine-N-carbonyl chloride; on the uppel right, beulzoyl L-phenylalanine methyl ester; on the lower right, D-CDIC. Asterisks indicate the target carbonyl carbons of the carboxylic acid derivatives; the leaving group, whether chloride or ester, is marked X. As noted above, the D-substrate is arranged so that its aromatic ring coincides with the benzoyl group (ring A) of benzoyl L- phenylalanine methyl ester. It should also be noted that the amide bonds coincide. The position of ring B could not be ascertained from models of benzoyl L-phenylalanine methyl ester and the D-substrate alone. The rigidity of PCC, however, allows us to position ring B in space. Its plane is at about 900 to that of ring A. The susceptible carbonyl carbons (asterisks) are attacked by the enzyme from below. As required, their positions in space coincide in the three models. Downloaded by guest on September 26, 2021 706J BIOCHEMISTRY: B. F. ERLANGER PROC. N. A. S. FiG. 2.-Models of phenothiazine-N-carbonyl chlor- FIG. 3.-Models of N-phenyl- ide (upper left), benzoyl-Lphenylalanine methyl ester benzohydroxamic acid and benzoyl- (upper right), and D-l-keto-3-carbomethoxy-1,2,3,4- L-phenylalanine methyl ester (X tetrahydroisoquinoline (lower right). Leaving group serving to indicate ester leaving indicated by X; acylamido oxygen, OI; carboxyl group). Arrow indicates carboxyl oxygen, Oli. Enzyme makes attack from below on carbon which is in position that carboxyl carbon marked with asterisk. would be occupied by phosphorus atom of inactivated chymotrypsin. Asterisk designates nucleophilic oxygen of hydroxamic acid. Other designations similar to those in Fig. 2. In Figure 3 are models of benzoyl L-phenylalanine methyl ester (upper) and N phenylbenzohydroxamic acid (lower).

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