Part 2:Mutational analysis reinforces trimer formation essential for enzyme activity
The structure of AcpS have been shown to be relatively conserved. All known p-pant transferase have a trimer conformation[ 4 ]. Burying of the large hydrophobic surface of the A-sheet seems to drive this trimer conformation. The three N termini of the trimer are located on one end of the  barrel and these termini contain a conserved motif ([V/I]G[V/I]D[V/I]; in single-letter amino acid code).This conserved stretch forms the first  strand, beta- 1, which packs against beta -4 and beta-5 from another molecule in the trimer[ 4 ]. The residues exposed towards the interior of this beta- barrel are largely hydrophobic. Ile5, one of the conserved residues, makes extensive contacts with residues His100, Val101, Ser102, Val115, Val114 and Gln113 of the neighboring molecule. In addition, Gln113, residue in the center of beta- strand  5, is the only polar residue buried in the interior of the  barrel[ 4 ]. Gln113 makes a tight hydrogen-bonding network with the Gln113 residues from the other two molecules in the trimer. Because neither of these amino acids are directly involved in CoA or ACP binding,  mutational effect on AcpS  may be primarily due to the packing of the trimer[ 4 ].
Various mutations have been studied such as mutation Gln113 >Glu,  results in a substitution of one amino acid by another with similar side chain size but different hydrogen bonding capacity. A Gln113> Arg substitution gives rise to a side chain with different size and polarity[ 4 ]. It was hypothesized that a non-conservative change such as Ile5> Arg would probably be required to disrupt the stable trimer structure[4]. Therefore Ile2 >Ala mutation, which resulted in a protein with a different retention time on the gel-filtration column, was selected for further studies[ 4 ].
The Ile2 >Ala, Gln113> Glu, Gln>113 Arg and Ile5 >Arg mutant proteins were expressed in E. coli as soluble proteins, purified to near homogeneity and characterized for structure and enzyme kinetics[ 4 ]. Except for the Ile2> Ala mutant, each showed a reduced expression level when compared with the wild-type enzyme. The effects of these mutations on AcpS activity were investigated by measuring the AcpS catalyzed attachment of a 14C-labeled acetyl-P-pant group onto apo-ACP[ 2 , 5 ].The wild type enzyme showed kinetics comparable to E.coli AcpS[ 2 , 5  ] . The wild-type enzyme showed an initial velocity of 30 min-1, at an ACP concentration of 2 µM and a CoA concentration of 100 µM . The effect of the mutation at position 113 has been shown to be  highly sensitive to the nature of the substituted amino acid. Of the various mutations tried, the initial velocity for the Gln113> Glu mutation was approximately sevenfold slower than the wild-type enzyme, Gln113 >Arg mutant was completely inactive and Ile5 >Arg mutant was also inactive. Ile2 >Ala mutant retained only 6% of the wild-type activity, even though the mutation is at the very N terminus of the polypeptide chain [ 4 ].
 Mutants with reduced molecular weight  do not show measurable activity suggesting one of three possibilities: that the trimer is necessary for activity; structure around th active site of the protein gets disrupted due to mutation; or that these single-point mutations lead to misfolded proteins[ 4 ]. However since all the mutants had similar retention time on ion exchange columns and NMR spectra analysis of isotopically labeled Ile2> Ala AcpS  indicated that it has a well-defined tertiary structure (GY Xu, unpublished observations) the last possibility is ruled out[ 4 ]. The reduced molecular weights of these mutants suggest that the wild-type trimer is the only catalytically competent form. The molecular weight of Gln113> Glu mutant is consistent with a trimer at the high concentrations used in the size-exclusion experiment. The fact that it retained some activity and its high molecular weight suggests the possibility of an alternate hydrogen-bonding network for Glu113 that allows for the formation of the trimer[ 4 ]. This mutation could be interpreted as shifting the equilibrium away from the active trimer, resulting in reduced activity. The final mutation, Ile2 >Ala, can also have the same interpretation, though its proximity to Asp8, one of the ligands to the metal in the active site, and may lead to local structural perturbation as a mode of inhibition[ 4 ].