Inhibition of estrogen binding to rat alpha-fetoprotein by tryptophan p-nitrophenyl esters

Rat alpha-fetoprotein (AFP) contains a site that both binds the protease substrate tryptophan methyl ester (TrpOMe) and influences estrogen binding. We have studied the effect of changing the amino acid and ester portions of this compound on its binding to AFP, as measured by the ability of the ester to inhibit binding of [3H]-estrone to AFP. We find that: (1) AFP binds tryptophan esters better than phenylalanine or tyrosine esters, (2) substitution of butyl or benzyl for the methyl group in TrpOMe increases binding for AFP by 30- to 100-fold, (3) substitution of p-nitrophenol at the ester position increases binding for AFP by 10(5), (4) p-nitrophenyl substitution in the ester position of tyrosine or phenylalanine methyl ester increases the affinity for AFP by 10(3) to 10(4), (5) inhibition of estrogen binding to AFP by tryptophan p-nitrophenyl esters is reversible and competitive, and (6) the hydrolysis products of tryptophan p-nitrophenyl ester are ineffective in inhibiting estrogen binding to AFP. Based on these results, we suggest that AFP contains a tryptophan ester recognition site which binds p-nitrophenyl esters with high affinity and influences estrogen binding. The ester binding site may be located spatially near the estrogen binding site.