AMDase

Although the absolute configurations of the products are opposite to that of antiinflammatory active compounds, and the substrate specificity is rather restricted as to the steric bulkiness around the reaction center, the enzyme system of A. bronchisepticus was proved to have a unique reactivity. Thus, detailed studies on the isolated enzyme were expected to elucidate some new interesting mechanism of the new type of decarboxylation. Thus, the enzyme was purified. (The enzyme is now registered as EC 4.1.1.76.) The molecular mass was about 24kDa. The enzyme was named as arylmalonate decarboxylase (AMDase), as the rate of the decarboxylation of phenylmalonic acid was faster than that of the a-methyl derivative. ... 

To clarify the characteristics of AMDase, the effects of some additives were examined using phenylmalonic acid as the representative substrate. The addihon of ATP and coenzyme A did not enhance the rate of the reaction, different from the case of malonyl-CoA decarboxylase and others in those, ATP and substrate acid form a mixed anhydride, which in turn reacts with coenzyme A to form a thiol ester of the substrate. In the present case, as both ATP and CoA-SH had no effect, the mechanism of the reaction will be totally different from the ordinary one described above. It is well estabhshed that avidin is a potent inhibitor of the formation of the biotin-enzyme complex. In the case of AMDase, addition of avidin has no influence on the enzyme activity, indicating that AMDase is not a biotin enzyme. 

Thus, AMDase requires no cofactors and this fact is entirely different from those of known analogous enzymes, such as acyl-CoA carboxylases, methylmalonyl-CoA decarboxylases " and transcarboxylases. 

For more detailed studies on this unique enzyme, the gene of AMDase was cloned using the direct expression method. The gene was clarified to be consisting of 720 bp, indicating that the enzyme consists of 240 amino acids (Fig. 9). 

Figure 9 Nucleotide and deduced amino acid sequences of AMDase.

Figure 10 Hammett plot of the of the AMDase-catalyzed decarboxylation of...

DNA sequence indicated that AMDase contains four cysteine residues located at 101, 148, 171 and 188 from amino terminal (Eig. 9). At least one of these four is estimated to play an essential role in the decarboxylation. The most effective way to determine which Cys is responsible to enzyme activity will be site-directed mutagenesis. To determine which amino acid should be introduced in place of active Cys, its role was estimated as illustrated in Eig. 13. One possibility is that... 

However, this is not so easy without the tertiary structure of the enzyme. The possible clues are the homology search with functionally resembling enzymes and computer simulation of the tert-structure of the enzyme. The characteristic features of AMDase are (i) the reaction proceeds via an enolate-type transition state, (ii) the cysteine residue plays an essential role and (iii) the reaction involves an inversion of configuration on the a-carbon of the carboxyl group. 

Figure 14 Homology alignment of active site of AMDase and some isomerases.

The Gly74Cys mutant was prepared via PCR using the plasmid that contains the gene-coding native AMDase. Although the change in amino acid is drastic, the mutant still exhibited some activity. As expected, the products were nearly racemic, if not entirely, in the case of the two substrates mentioned above. These results demonstrate that this position is effective to give a proton to the intermediate of the reaction. 

The molecular mass of the native AMDase was estimated to be about 22 kDa by gel filtration on HPLC. Determination of the molecular mass of denatured protein by SDS-PAGE gave a value of 24 kDa. These results indicate that the purified enzyme is a monomeric protein. The enzyme had an isoelectric point of 4.7. The amino acid sequence of the NH2-terminus of the enzyme was determined to be Metl-Gln-Gln-Ala-Ser5-Thr-Pro-Thr-Ile-Glyl0-Met-Ile-Val-Pro-Prol5-Ala-Ala-Gly-Leu-Val20-Pro-Ala-Asp-Gly-Ala25. 

Table 2. Purification Table of AMDase from A. bronchisepticus...

Table 3. Synthesis of optically active a-arylpropionates using AMDase" ...

Fig. 3. Nucleoside sequence of the DNA fragment containing the AMDase gene...

The DNA sequence of the encoding AMDase and the amino acid sequence deduced from it was compared with the data base using DNASIS (Hitachi). No significant homologies were observed with any of the sequences searched. 

In the previous studies using inhibitors and additives, it became clear that AMDase requires no cofactors, such as biotin, coenzyme A and ATP. It is also suggested that at least one of four cysteine residues plays an essential role in asymmetric decarboxylation. One possibility is that the free SH group of a cysteine residue activates the substrate in place of coenzyme A. Aiming at an approach to the mechanism of the new reaction, an active site-directed inhibitor was screened and its mode of interaction was studied. Also, site-directed mutagenesis of the gene coding the enzyme was performed in order to determine which Cys is located in the active site. 

We screened for a potent inhibitor against the AMDase-catalyzed decarboxylation of a-methyl-a-phenylmalonic acid to give a-phenylpropionic acid. Among the compounds shown in Fig. 4 which have structures similar to the substrate. 

Fig. 5. Inhibition mode of a-bromophenylacetic acid against AMDase-catalyzed decarboxylation. Lineweaver-Burk plot in the presence of the acid A, 100 pM B, 20 pM C, 0 pM...

As deduced from the DNA sequence of the gene, AMDase contains four cysteine residues. Since a-halocarboxylic acids are generally active alkylating agents there is a possibility that a-bromophenylacetic acid reacts with several cysteine residues of the enzyme. Therefore, we tried to clarify how many cysteine residues react with this inhibitor. It is well established that when p-chloromercuri-benzoate (PCMB) binds to a cysteine residue, the absorbance at 255 nm increases due to the formation of an aryl-Hg-S bond. Thus it is possible to estimate the number of free S-H residues of the enzyme by titration with PCMB solution (Fig. 6). When the native enzyme had reacted with PCMB, the absorbance at 255 nm increased by 0.025. On the other hand, when PCMB solution was added to the enzyme solution after the enzyme was incubated with a-bromophenyl-... 

There are at least three possibile ways in which the inhibitor can bind to the active site (1) formation of a sulfide bond to a cysteine residue, with elimination of hydrogen bromide [Eq. (10)], (2) formation of a thiol ester bond with a cysteine residue at the active site [Eq. (11)], and (3) formation of a salt between the carboxyl group of the inhibitor and some basic side chain of the enzyme [Eq. (12)]. To distinguish between these three possibilities, the mass numbers of the enzyme and enzyme-inhibitor complex were measured with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI). The mass number of the native AMDase was observed as 24766, which is in good agreement with the calculated value, 24734. An aqueous solution of a-bromo-phenylacetic acid was added to the enzyme solution, and the mass spectrum of the complex was measured after 10 minutes. The peak is observed at mass number 24967. If the inhibitor and the enzyme bind to form a sulfide with elimination of HBr, the mass number should be 24868, which is smaller by about one... 

Site-directed mutagenesis is one of the most powerful methods of studying mechanisms of enzyme-catalyzed reactions. Since this technique makes it possible to replace a specific amino acid residue of an enzyme by an arbitrary one, it is particularly useful to specify the amino acid residue(s) which is responsible for the activity [20 - 22]. In the case of AMDase, one of four cysteine residues was presumed to be involved in the catalytic site by the titration experiments. To determine which Cys is located at the active site, preparation of four mutant enzymes, in each of which one of the cysteines is replaced another amino acid, and kinetic studies on them, are expected to be most informative. Which amino acid should be introduced in place of cysteine To decide on the best candidate. 

Table 6. Kinetic constants of AMDase for substituted phenylmalonic acis ...

Fig. 7. Hammett plot of k at for the AMDase catalyzed decarboxylation of a series of X-phenyl-malonic acids...

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