E speculate that our purification protocol, which involved incubating purified PepN with an excess of Zn(II), resulted in Zn(II) occupancy of among these web pages. The specificity profile of each PepN variant was determined having a panel of six X-Ala substrates (Fig. 4A). Substitution at Met-260 resulted in substantial adjustments in enzyme specificity. Interestingly, the M260P substitution recapitulated the specificity for straight-chain P1 side chains that was observed with PfA-M1 V459P. Catalytic efficiencies for PfA-M1 and PepN variants bearing identical configurations of S1 cylinder residues are compared in Fig. 4B. Generally, PepN variants have been far better catalysts with P1-Arg and -Lys substrates than their PfA-M1 counterparts. Differences in S1 cap residues inside the two enzymes, that are positioned in the major on the S1 subsite and in PepN interact with P1-Arg and -Lys side chains (13), may well be responsible for these specificity variations (see “Discussion”). Structural Basis for the Restricted Specificity of PfA-M1 V459P–We were intrigued by the marked shift in specificity to P1-Arg, -Lys, and -Met that was effected by replacement of theJOURNAL OF BIOLOGICAL CHEMISTRYM1-aminopeptidase SpecificityFIGURE two. Effects of substitution at residue 459 in PfA-M1 on the steady-state kinetic parameters Km and kcat. A, Km values are represented with blue bars along with the scale at left and kcat values with red bars as well as the scale at suitable. The substrate is indicated within the upper appropriate of every panel. The identity on the residue at position 459 is indicated on the abscissa (wild-type V). B, -fold adjust of Km and kcat values for each of the 4 dipeptide substrates is expressed as the ratio on the maximal value for the minimal value. C, plots of kcat versus Km for Phe-Ala, Leu-Ala, Ala-Ala, and Arg-Ala for PfA-M1 variants with nonpolar residues at position 459 (Gly, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp) are shown. The square with the Pearson correlation coefficient (R2) is reported for each linear regression fit to indicate the extent with the correlation amongst kcat and Km.variable S1 cylinder residue with proline in PfA-M1 and PepN. To decide the structural basis for this phenomenon, we solved the crystal structure of PfA-M1 V459P in complicated with a molecule of arginine to 2.two ?resolution (Table 2). Alignment of the structures of wild-type, unliganded PfA-M1 ((21), PDB 3EBG) and PfA-M1 V459P-Arg yielded an 0.4 ?root imply square deviation for backbone atoms. Along with the V459P mutation, we noticed that the residue His-378 within the our structure is replaced with proline within the PfA-M1 structures reported by McGowan et al. (21). This His-to-Pro transform at residue 378 brought on a repositioning in the backbone of residues 376 ?79 having a maximal displacement of two.5,7-Dibromoquinoline Chemical name 1 ?for the -carbon of residue 377 (data not shown).883-40-9 web The genome sequence with the 3D7 clone of P.PMID:23903683 falciparum (28), which was the source with the PfA-M1 sequence made use of within this study, indicates that a histidine is encoded at residue 378 within this strain. The active web page of PfA-M1 V459P was occupied by a single arginine molecule (Fig. 5, A and B). The amino and carboxylate groups on the Arg ligand formed multiple interactions together with the enzyme, plus the guanidinium group interacted together with the carboxylate group on the S1 cap residue Glu-572. Comparison in the S1 subsite of unliganded wild-type PfA-M1 to that with the V459P-Arg structure revealed that the proline substitution brought on the polypeptide backbone to move towar.