The specificity of hippuricase
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The theoretical background for a proposed method for the separation of specific enzymes by methods related to those of immunochemistry is presented in the introduction. The formation of enzyme-substrate complexes during enzymic reactions and the theory that antibody-antigen and antibody-hapten group combinations are of the same type as the enzyme-substrate complex is discussed. A review of the available literature concerning the enzyme hippuricase (histozyme) is given. Much work has been done in the study of the specificity of the enzyme and its differentiation from enzymes splitting similar peptide linkages. The most general statement that can be made as to the type of compound hydrolyzed by the enzyme is that acyl derivatives of amino acids are hydrolyzed. However, there is a feeling by some of the workers that several specific enzymes are present in the preparations used and that the individual enzymes are rather specific for both the acyl and the amino acid group. The literature was found to be entirely lacking in studies of the effect of ring substituents on the hydrolysis of hippuric acid by hippuricase. Isomeric monosubstituted hippuric acids were prepared. These included the ortho-, meta- and para- isomers of methylhippuric, chlorohippuric, bromohippuric, iodohippuric, nitrohippuric acids and m-aminohippuric acid. In most cases the benzoic acids necessary for the synthesis of the corresponding hippuric acids were prepared by converting the respective toluidine to monosubstituted benzoic acids by well established series of reactions. Some related compounds were also prepared for testing against the enzyme. The amount of hydrolysis of the hippuric acids in the presence of hippuricase was determined by Sorensen formal titrations. Staphylococcus aureus(strain B985) showed significant hippuricase activity. One-half ml. of a day old culture of the bacteria in 100 ml. of a solution 0.02 M in neutralized hippuric acid hydrolyzed 70% of the substrate. The enzyme preparation used in the experiments on hippuricase was Taka-Diastase. The preparation, which was standardized to liquify 450 times its weight of starch in ten minutes, was found to be satisfactory. The rate of hydrolysis of an 0.02 M solution of hippuric acid neutralized with 0.1 N NaOH was found to be approximately proportional to the enzyme concentration in the range of 1 to 4 g.% of Taka-Diastase. Using 4.g.% T aka-Diastase, it was found that the amount of hippuric hydrolyzed per unit of time was constant between 0.02 and 0.1 Molar. Thus, the reaction was zero order in this range of substrate concentration. The studies of the enzyme and substrate concentration showed that a four gram-percent concentration of T aka-Diastase and 0.02 M substrate would give the desired result with hippuric acid as the substrate. These concentrations were strictly adhered to in the experiments that follow. In order to determine the effect of substituents in the benzene ring of hippuric acid in reference to the activity of hippuricase on the peptide linkage, methyl-, chloro-, bromo-, iodo-, nitro- and amino- hippuric acids were tested against the enzyme. In studying the hydrolysis of the isomeric chlorohippuric acids by the enzyme hippuricase, it was found that the ortho-chlorohippuric acid was unattacked. The meta-derivative was hydrolyzed at a rather rapid rate and the para-chlorohippuric acid was hydrolyz.ed to a lesser extent than the meta. The amount of hydrolysis in two days for the three substrates were: ortho, O%; meta, 19%; para, 11%. A study was made of the activity of hippuricase on the hydrolysis of the ortho-, meta- and para-bromohippuric acids at several intervals over a period of seven days. Under the conditions of the tests ortho-bromohippuric acid was hydrolyzed at a very slow rate, the para-derivative at moderate rate and meta-bromohippuric acid extremely rapidly. The following were the amounts of hydrolysis at the end of the seven day period: ortho, 6%; meta, 98%; para, 39%. The study of the series of iodo- derivatives of hippuric acid gave results similar to those in the previous experiments with the bromohippuric acids. Thus, at the end of a seven day period the total amounts of hydrolysis were the following: ortho, 2%; meta, 62%; para, 31%. Isomeric nitrohippuric acids showed essentially the same rates of attack by hippuricase as the halogen derivatives of hippuric acid. The following were the results of the hydrolysis of the nitrohippuric acids over a period of seven days: ortho, O%; meta, 89%; para, 20%. The action of hippuricase on the methylhippuric acids was similar to its action on the other derivatives of hippuric acid. The amounts of hydrolysis of each isomer occurring in two days were: ortho, O%; meta, 37%; para, 26%. Meta-aminohippuric acid was hydrolized to an extent of 4% in two days. From these results we can conclude that substitution in the ortho- position of hippuric acid inhibits the hydrolytic action of hippuricase almost completely. Substitution in the para- position causes about 50% inhibition. A group substituted in the meta- position has no inhibitory action on the hydrolysis by hippuricase and may cause a stimulatory effect. Hippuricase can hydrolyze the acetamidobenzoic acids. The effect on these compounds appears to be different from the effect on the hippuric acid derivatives. In the experiments with these compounds, the meta- isomer was hydrolyzed to the least extent, the para- next and the ortho- derivative most. The amounts of hydrolysis occurring in ten days were: ortho, 36.5%; meta, 6%; para, 20%. The acetotoluidides were unattacked by the enzyme. Thus it appeared that the free carboxylic group was necessary for the compound to be attacked by hippuricase. In order to determine the ability of hippuricase to attack the carbamyl (-CONH2) group, compounds containing this group attached to organic radicals were tested. The compounds tested were benzarnide, acetamide, urea and urethane. A related compound, creatine, was also tested. Though creatine does not contain a true carbamyl group, it does possess a closely related group and also a free carboxyl group. None of these compounds were attacked by the enzyme. Thus it was determined that hippuricase is not an amidase. Other compounds that were found to be unattacked by the enzyme were acetanilide, acetphenetidine and benzanilide. These negative results tend to show that hippuricase cannot be called a general acylase. The action of the enzyme on the individual bile acids showed a slight but significant hydrolysis of glycocholic acid and an almost insignificant hydrolysis of taurocholic acid. There was found to be no hydrolysis of benzenesulfonylglycine in the presence of hippuricase.
Thesis (Ph.D.)--Boston University
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