KATII was previously indicated to be an ADD aminotransferase. Indeed, the catalytic efficiency of the enzyme towards AAD is slightly higher than that towards KYN . The reaction of KATII with AAD in the absence of ketoacids leads to the transamination of PLP to form PMP (Fig. 1A), which accumulates in the presence of KG. Thus, at equilibrium, the PMP form of KATII is the most stable enzyme form; that is, the rate of its formation is higher than the rate of its consumption. The high absorbance of KYN in the spectral range where PLP and PMP intermediates also absorb hampers performance of the same analysis for the reaction between KYN, KG, and KATII.
PLP-dependent enzymes are reported to be quite promiscuous, with respect to both substrate and the types of reaction catalyzed. It is known that many PLP-dependent enzymes, including tryptophan synthase , 3,4-dihydroxyphenylalanine decarboxylase , AspAT , and serine racemase [107,108], can catalyze side reactions, some of which have been suggested to play physiological roles [107,108]. In particular, aminotransferases are known to be (relatively) efficient in catalyzing β-elimination reactions, especially on substrates with good leaving groups, such as BCA and, more interestingly, on S-substituted cysteine derivatives [42,43,72,73,85,109,110]. In the case of KATII, the exploration of β-lyase activity was particularly intriguing, in that structural comparisons suggested the presence of features normally observed in β-lyases, such as the conformation of the N-terminal region and a tyrosine at position 142, which, in most aminotransferases, is occupied by a tryptophan or a phenylalanine . Thus, we investigated the β-lytic activity of KATII and Tyr142→Phe KATII with BCA and SPC. Whereas the Tyr142→Phe mutation does not change the reactivity of KATII towards AAD and KYN in the presence of KG, it heavily influences the β-lytic activity of the enzyme. In fact, both the wild type and the mutant are able to eliminate chloride from BCA, with the production of ammonia and pyruvate, but the Tyr142→Phe mutant is 20-fold less efficient. One possible explanation is that the tyrosine at position 142 plays a role in the balance between β-elimination and transamination. In the conditions tested here, we could not reach saturation in a plot of initial velocity against BCA concentration. This is consistent with the observation that Km values of transaminases for BCA are usually very high, preventing the determination of kinetic parameters. This also hampers the calculation of catalytic efficiency for chloride elimination by KATII and Tyr142→Phe KATII. For this reason, we cannot rule out the possibility that the lower rate of β-elimination observed for Tyr142→Phe KATII is partly attributable to a higher Km value. Although a detailed characterization of the β-lytic activity of this mutant is outside the scope of this work, we checked the β-lytic activity of wild-type KATII and the mutant enzyme in the presence of SPC. Also in this case, we observed β-elimination with production of ammonia by the wild-type enzyme but no activity of the Tyr142→Phe mutant, a further indication of a reduction of β-lytic activity brought about by the mutation. Interestingly, the β-elimination, although with a very low ratio with respect to transamination, also takes place on the natural substrate KYN. This unusual β-elimination reaction should produce o-aminobenzaldehyde, as already reported in a controversial paper on the activity of kynureninase . At present, it is not known whether this reaction has any physiological significance or is regulated by any effector, as is the case, for example, with the mammalian serine racemase [107,108]. However, one should bear in mind that the β-elimination reaction requires the formation of an α-aminoacrylate intermediate that, in the case of KATII, as demonstrated by experiments with BCA, leads to concomitant syncatalytic inactivation of the enzyme. Furthermore, it is unlikely that a very inefficient reaction, when compared to the main one, would have any physiological significance, unless it is tuned by effectors and ligands. The understanding of this aspect of the KATII mechanism of action is beyond the aim of this work, but deserves further attention.
As expected on the basis of the ESBA structure, KATII catalyzes both transamination and β-elimination of this compound. A rough estimate of the catalytic efficiency of the β-elimination reaction, based on specific activity at a fixed substrate concentration, indicates that ESBA, like KYN, is a poor substrate for β-elimination, when compared with BCA. However, both ESBA and BCA are capable of permanently inactivating KATII through a mechanism that probably involves the formation of a covalent adduct between the active site lysine and the α-aminoacrylate intermediate, as already reported for alanine aminotransferase (AlaAT)  and AspAT  (Scheme 4). We were unable to recover a PLP–ESBA derivative, either after ultrafiltration or after gel filtration on microspin columns (data not shown). This hampered the determination of the type of modification by MS. The dependence of the percentage of initial activity of KATII on time of incubation in the presence of 5 mm BCA (data not shown) gives an exponential decay with kobs = 0.7 min−1 (t1/2 ∼ 1 min). In the case of AlaAT, the pseudo-first-order rate constant for inactivation was 0.36 min−1, with t1/2 ∼ 2 min, in the presence of 5 mm BCA . The partition ratio (moles of product per mole of inactivated enzyme) is about 500, a value comparable with those found for AlaAT, 1050  and for kynureninase, 530 .