Studies on receptor binding site of insulin: The hydrophobic B12Val can be substituted by hydrophilic Thr

Summary: [B12Thr]human insulin and [Bl2Leu]human insulin were obtained by means of site-directed random mutagenesis. [B12Thr]human insulin retains total biological activity but [B 12Leu]human insulin has much lower biological activity. Receptor binding activities of [B 12Thr]human insulin and [B 12Leu]human insulin are 56% and 3%, respectively, as that of native porcine insulin. The results suggest that the hydrophobic property of the residue side chain at B12 may not be necessary. The three-dimensional structure of insulin shows that in the monomer the side chains ofB12Val, B16Tyr, B24Phe and B25Phe form a hydrophobic surface [12]. This hydrophobic surface was proposed to be a key constituent part of the receptor binding site of insulin[3-6]. It is interesting to study the role of B 12Val in the structure and function of insulin since it is a constituent residue of the hydrophobic surface and it is also conserved in insulins from different species[7]. Schwartz and Hu reported that insulin analogs, in which BI2Val was substituted by Asn, Phe or cz-aminoisobutyric acid by means of chemical synthesis, had much lower biological activities than native insulin [8-9]. Similarly when B 12Val was substituted by Ile or Glu, the biological activity of these analogs was greatly reduced [10]. For further understanding the role of the residue at position B 12 in the receptor binding site of insulin, we have prepared, by means of sitedirected random mtagensis, two mutants of porcine insulin precursor(PIP) in