NUCLEOTIDE-BINDING BY MULTIENZYME PEPTIDE SYNTHETASES

Peptide synthetases consist of linearly arranged catalytic units, whic h by sequence alignment show equally spaced amino-acid-activating segm ents/modules of 600-700 amino acid residues. The consensus sequence co mprises a new class of sequence motifs which are shared by some carbox yl-activating enzymes, but which do not occur in aminoacyl-tRNA synthe tases. The catalytic properties of peptide synthetases with respect to the nucleotide substrate were investigated by enzyme kinetic studies. In the activation reaction ATP may be substituted by 2'-deoxy-ATP (dA TP) and 7-deazaadenosine 5'-triphosphate, substrate analogues which ar e not recognised by many aminoacyl-tRNA synthetases, and may thus prov e useful alternative substrates in the detection of peptide synthetase s within complex protein mixtures. ATP derivatives substituted at C2 a re substrates, while those substituted at C8 are not, indicating a pre ference for the anti-conformation in substrate binding. Kinetic studie s revealed that coenzyme A is a non-competitive inhibitor of the activ ation reaction, suggesting the presence of a second nucleotide binding site which accommodates nucleotides with phosphate in the C2' or C3' position. This substrate and inhibition profile is markedly different from that of aminoacyl-tRNA synthetases and indicative of a separate h omogeneous family of carboxyl-activating enzymes.