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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

A3 Val is important for insulin activity. It is invariant in insulins from different species studied thus far. Based on the three dimensional structure of insulin, it was thought to be involved in receptor binding. Its replacement by Leu resulted in remarkable lowering of insulin activity, indicating the crucial requirement of the side chain geometry at this position. When A3 Val was replaced by Thr, which is hydrophilic but isosteric with Val, substantial insulin activity was retained. Therefore, the isosteric requirement for receptor binding at this site is more stringent than the hydrophobic requirement. Here we report the replacement of A3 Val of porcine insulin by the unnatural allo-Thr. The in vivo biological activity of A3 allo-Thr insulin is similar to that of A3 Thr insulin or native insulin, but its receptor binding activity is 7.6% instead of 50% for A3 Thr insulin, indicating that at the A3 position the hydrophilic OH group of Thr could be more tolerated in receptor binding than the OH group of allo-Thr. The retention of insulin activity by substituting A3 Val with the unnatural isosteric allo-Thr demonstrates again the importance of isosteric interaction in the binding of insulin with its receptor.

Single-chain insulin/IGF-1 hybrid-[Ins/IGF-1(C)], single-chain porcine insulin precursor-(PIP), and B10Asp PIP were prepared by protein engineering. Their growth-promoting activities in mouse breast cancer cell line GR2H6 are 10, 0.2, and 2 times that of insulin, respectively, and 29%, 0.6%, and 6% of that of IGF-1, indicating that the C domain and 9Glu of IGF-1 are important for its growth-promoting activity. Given these results and previous reports, we propose that the C domain, 9Glu, and 23Phe-26Asn beta bend are involved in the growth-promoting functional region of IGF-1.