 acceptor from the downstream PCP domain a situation which is not given in the cyclization experiment with hexapeptidyl-holo-pcp-c TycC5-6. One possibility for the observed cyclization was that it may not be actively catalyzed by the C domain. Instead, it could be a side effect of either the loading onto the PCP domain or the altered environment altogether. To test this, the hexapeptide was loaded onto the isolated PCP TycC6, which led to no formation of a cyclic or even hydrolytic product under otherwise identical conditions. A second test was performed by using a mutant of PCP-C TycC5-6, in which the conserved, supposedly catalytic histidine residue (H224) [Bergendahl 2002, Roche 2003] had been exchanged by alanine in one case and valine in the other. Both resulting proteins were well-soluble and their α-helical CD spectra were in good 90
91 6. Discussion agreement with the wildtype enzyme. When these mutants were used in analogous cyclization experiments, no cyclized hexapeptide was produced. Further investigations with variations of the DPhe-Pro-Phe-DPhe-Asn-Gln substrate revealed that Gln6 could not be replaced by alanine at the C-terminus and a D-configured N-terminal amino acid is necessary for cyclization to take place. When the chain length was increased to 8 by insertion of two alanines after position 4, the linear substrate was only released by C-terminal hydrolysis. Unfortunately, substrates with a C-terminal ornithine (as in the natural substrate) were not accessible due to instant cyclization of the peptide moiety in the corresponding CoA species, likely mediated by the intramolecular attack of the free δ-amino group onto the thioester, which leads to a six-membered lactam. However, glutamine appeared similar enough, as its only difference to ornithine is the carbonyl oxygen at the γ-position which abolishes most of the nucleophilicity of the δ- amino group since the latter is now part of a mesomerically stabilized amide with a partial positive charge on the nitrogen atom. The fact that DPhe1 could not be replaced by LPhe or LAla, on the other hand, was more puzzling, because the natural acceptor substrate leucine is L-configured. For the cyclization of a hexapeptide in a head-to-tail manner, a pre-folding of the linear precursor must be possible to allow for a proximal orientation of both termini. It can be assumed, that the delicate sequence of L- and D-configured residues especially, when sterically demanding such as phenylalanine can have a dramatic effect on such a pre-folding. For the tyrocidine linear decapeptide a β-sheet like folding has been reported [Bu 2002], and following the 4n+2-rule, an analogous secondary structure can easily be envisioned for the hexapeptide, too. Thus, obviously both the C domain s catalytic residue H224 and an intrinsic effect of the substrate itself contribute to the observed phenomenon. These findings led to a deeper scrutiny of the previously suggested catalytic models of the C domain. The first one states that the second histidine of the core 3 motif HHxxxDG (this will be referred to as H224 like in PCP-C TycC5-6, to avoid confusion) acts as a base catalyst required to increase nucleophilicity of the acceptor α-amino group by either capturing the excess proton after the initial attack or the abstraction of a proton from the corresponding α-ammonium group before the attack onto the thioester group. 91
92 6. Discussion For C TycB1 it was experimentally shown by mutational studies that H224 is essential for catalytic activity [Bergendahl 2002], and analogous findings were made here in the context of the cyclization reaction with PCP-C TycC5-6. However, Keating and coworkers made contrary observations when testing the stand-alone C domain VibH [Keating 2002]. This C domain condenses PCP-bound dihydroxy-benzoate with the primary amino group of free norspermidine as part of the vibriobactin nonribosomal biosynthetic process in Vibrio cholerae. When H224 was mutated to alanine, merely a 90% decrease in activity was observed indicative of a non-essential role of the respective residue. It is argued that solely the positioning of both reactants by the C domain leads to the formation of the thermodynamically more stable amide. In the bidomain structure of PCP-C TycC5-6, a buffer-derived sulfate ion is seen in proximity to H224 (N ε2, 2.87 Å). The pk calculation for the side chain of H224 (pk 11.8) indicates that it is protonated in this environment (H++ server, [Bashford 1990, Gordon 2005]). Thus, it cannot act as Brønsted-base to perform catalysis. Instead, the binding of sulfate in this position suggests an electrostatic interaction between the negatively charged sulfate and the imidazolium group. Taking a closer look at the active site, two additional features are found that stabilize the sulfate ion in its very position (Figure 6.8): A hydrogen bond with the amide nitrogen of G229 and the dipole moment of helix 4. Figure 6.8: The chemical environment at the C domain s active site as seen in the bidomain structure. A sulfate ion is bound to H224 and G229 by polar interaction/hydrogen bonding. The dipole moment of helix 4 further stabilizes the sulfate. H223 is turned away from the substrate (solvent) channel. 92
93 6. Discussion The first histidine in the core 3 motiv HHxxxDG is found to be embedded in a hydrophobic pocket that is turned away from the substrate channel, making an involvement in the catalytic process rather unlikely, unless substantial structural re-arrangements take place. Accordingly, its calculated pk value of -3 is very low. With these findings, a different catalytic model for the C domain can be suggested here if one assumption is made: The negatively charged, tetrahedral sulfate ion found in the crystal is analogous to the transition state of the amide bond forming reaction in the C domain. This transition state generated by the nucleophilic attack of the α-amino group on the thioester carbon atom is also sp 3 -configured and carries a negative charge (oxyanion). The structural features and interactions that can be held accountable for the binding of sulfate in the crystal can in turn be pictured as the enzymatic environment that stabilizes the transition state. As a result, the activation energy is lowered and the reaction speed increased. This model does not contradict the previous findings that H224 is essential for TycB1 and TycC6 while it is not for VibH. The fact that three interactions with the enzyme separately contribute to the transition state stabilization leaves room for the possibility that in individual cases only two of them might be sufficient. One reason for the postulate of a Brønsted-base in the catalytic site of the C domain originates from the concept that primary amines are expected to be practially quantitatively protonated under physiological conditions and therefore cannot act as nucleophiles. When taking a closer look at several pk values, however, it becomes evident that this argument can be refuted. While aliphatic primary amines have pk values between 9.2 and 10.6 [Hall 1957], the α-amino groups of oligopeptides are less basic (pk Gly-Gly = 8.31, pk Ala-Ala-Ala = 8.03), and although values for aminoacyl thioesters could not be found in the literature, the pk value of glycine oxoester is reported to be 7.75 [Hall 1957]. Still, the majority of amino groups are protonated, but the un-protonated portion is able to perform the condensation reaction without a base, and the fact that the reactant is withdrawn from the equilibrium drives the reaction. This is consistent with the known issue of C domains being very slow catalysts compared to A domains, for instance (~1:1000). The overall architecture of both structurally known C domains, C TycC6 and VibH, clearly shows that there is no classical deep cavity for substrate binding but rather a canyon-like 93
94 6. Discussion groove in which the substrates are positioned. The natural substrates for VibH (PCP-bound dihydroxy-benzoate and norspermidine) and C TycC6 (nonapeptide and leucine, both PCPbound) vary greatly in size and shape, yet the enzymatic environments for their positioning are analogous. It is therefore expected that the Ppan cofactor coordination with the enzyme along the solvent channel is critical for the positioning of the reactants. Unfortunately, these interactions are not seen in the apo-pcp-c TycC5-6, but a structural superposition of the carnitine acetyltransferase/coa complex [Hsiao 2004] with the C domain s N-terminal subdomain places the pantetheine moiety along the floor of the canyon in such a way that the thiol group almost coincides with the sulfate ion at the active site (Figure 6.9). Figure 6.9: The bidomain structure of PCP-C TycC5-6 with Ppan modeled into the solvent channel. The PCP domain is shown as cartoon (green), and the C domain s surface plot is colored by its charge (red = acidic, blue = basic). The bridging region has been reduced to a transparent cartoon (yellow) for clarity. The Ppan arm thiol group almost coincides with the sulfate ion bound to H224. In the inhibitory studies perfomed with