Series of substrates derivatives of peptide deformylase were systematically
synthesized and studied for their capacities to undergo hydrolysis. Data a
nalysis indicated the requirement for a hydrophobic first side chain and fo
r at least two main chain carbonyl groups in the substrate. For instance, F
o-Met-OCH3 and Fo-Nle-OCH3 were the minimal substrates of peptide deformyla
se obtained in this study, while positively charged Fo-Nle-ArgNH(2) was the
most efficient substrate (k(cat)/K-m = 4.5 x 10(5) M-1 . s(-1)). On the ba
sis of this knowledge, 3-mercapto-2-benzylpropanoylglycine (thiorphan), a k
nown inhibitor of thermolysin, could be predicted and further shown to inhi
bit the deformylation reaction. The inhibition by this compound was competi
tive and proved to depend on the hydrophobicity at the P-1' position. Spect
roscopic evidence that the sulfur group of thiorphan binds next to the acti
ve site metal ion on the enzyme could be obtained. Consequently, a small th
iopseudopeptide derived from Fo-Nle-OCH3 was designed and synthesized. This
compound behaved as a competitive inhibitor of peptide deformylase with K-
I = 52 +/- 5 mu M. Introduction of a positive charge to this thiopeptide vi
a addition of an arginine at P-2' improved the inhibition constant up to 2.
5 +/- 0.5 mu M, a value 4 orders of magnitude smaller than that of the star
ting inhibitors. Evidence that this inhibitor, imino [(5-methoxy-5-oxo-4-[[
2-(sulfanylmethyl)hexanoyl] amino]pentyl)amino]methanamine, binds inside th
e active site cavity of peptide deformylase, while keeping intact the 3D fo
ld of the protein, was provided by NMR. A fingerprint of the interaction of
the inhibitor with the residues of the enzyme was obtained.