To determine whether there were gross changes to the secondary structures of the mosaic PBPs, we analyzed the sPBPs by low-resolution CD spectroscopy to estimate the distribution of α-helical Galunisertib and β-sheet structures (Venyaminov & Yang, 1996; Sreerama et al., 1999). The predicted secondary structures indicated that there were no substantial differences among any of the sPBPs (Table 2), suggesting that their overall folding patterns remained intact. The results eliminated this
trivial explanation for the inability of PBP 6 and PBP 565 to complement shape defects in vivo. β-Lactam antibiotics bind covalently to a serine residue at the active site of PBPs, thereby inactivating the enzymes. Because β-lactams are substrate analogues of the d-alanyl-d-alanine terminus of the peptide side chain in peptidoglycan (Park & Strominger, 1957; Park, 1996), the rate of acylation by penicillin measures one facet of the enzymatic activity of the PBPs. To determine how efficiently sPBPs bound penicillin, we assessed the interaction of each sPBP with
BOCILLIN FL. The acylation rate (k2/K) for sPBP 5 was approximately 40% of the rate observed for sPBP 6 (Table 3). The rate for mosaic protein sPBP 656 was ∼70% of that for sPBP 6, which was >50% greater than that of sPBP 5. Thus, grafting the MMD of PBP 5 into PBP 6 decreased the penicillin acylation rate of sPBP 6, although the rate remained higher than that Ixazomib supplier of wild-type sPBP 5 (Table 3). This indicates that
the MMD of PBP 5 is important, but does not by itself determine the efficiency of acylation in the context of PBP 6. On the other hand, the acylation rate for sPBP 565 was drastically lower than that of PBP 5. Therefore, placing the MMD of PBP 6 in PBP 5 decreased the acylation rate of PBP 5 by 98% of its former value. To understand how efficiently the sPBPs released bound penicillin from the acyl–enzyme complex (a measure of the catalytic efficiency), k3 values were determined for each of the constructs. Reverse transcriptase The acylation rate for sPBP 6 was about 10 times less than that of sPBP 5 (Table 3). However, upon grafting the stretch of amino acids that corresponds to the MMD of PBP 5 into PBP 6 (i.e. sPBP 656), the deacylation efficiency of sPBP 6 increased fourfold. In contrast, the hydrolysis of BOCILLIN FL by sPBP 565 was too slow to measure under laboratory conditions, indicating that although the PBP 5 MMD was partially efficient in influencing deacylation of the BOCILLIN substrate, the corresponding stretch of amino acids from PBP 6 had no such effect. Taken together, the influence of the PBP 5 MMD on acylation and deacylation is noteworthy, and the rates of penicillin acylation or deacylation can serve as good predictors for the ability of PBPs 5 or 6 or of their mosaic counterparts to complement morphological defects of E. coli shape mutants.