Substitution of D-Trp32 in NPY destabilizes the binding transition state to the Y1 receptor site in SK-N-MC cell membranes.

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.