Figure 1
- Kinetic model of a protease-catalyzed acyl transfer reaction. EH, Free
enzyme; Ac-X, acyl donor; HX, leaving group; Ac-E, acyl enzyme complex; Ac-OH, hydrolysis
product; HN, acyl acceptor; Ac-N, aminolysis product.
Figure 2
- Schematic comparison of the binding of a peptide-4-guanidinophenyl ester and
a common peptide substrate to the active site of trypsin based on the ideas of the
conventional binding model of proteases (nomenclature according to Ref. 8).
Figure 3
- Correlation between the calculated binding energies of the most stable
productive complexes of Boc-Xaa-OGp and trypsin and the specificity constants of the
hydrolysis reaction (25). The correlation coefficients are R = -0.84 for the L-series
including Gly (triangles) and R = -0.91 (lozenges) for the D-series.
Figure 4
- Scheme of the extended kinetic model of a protease-catalyzed hydrolysis of
substrate mimetics introducing the novel rearrangement equilibrium constant KR
(25). EH, Free enzyme; Ac-X, substrate; [E..Ac-X], Michaelis-Menten complex; HX, leaving
group; E-Ac, acyl enzyme intermediate bearing the acyl residue at the S'-subsite; Ac-E,
acyl enzyme intermediate bearing the acyl residue at the S-subsite; Ac-OH, hydrolysis
product.
Figure 5
- Partition values p for the aminolysis of Boc-D/L-Ala-OGp and
Boc-D/L-Leu-OGp compared to the common substrate Bz-Arg-OEt by trypsin (25). Conditions:
0.2 M HEPES-buffer, pH 8.0, 0.2 M NaCl, 20 mM CaCl2, 25oC;
[substrate]: 2 mM, [amino component]: 20 mM; errors are less than 15%.
Figure 6
- Course of the clostripain-catalyzed (3+5) fragment condensation of
Boc-Phe-Gly-Gly-OGp and H-Ala-Phe-Ala-Ala-Gly-OH (27). Boc-Phe-Gly-Gly-OGp (triangles);
Boc-Phe-Gly-Gly-Ala-Phe-Ala-Ala-Gly-OH (squares); Boc-Phe-Gly-Gly-OH (circles).
Conditions: 50 mM HEPES-buffer, pH 8.0, 100 mM NaCl, 10 mM CaCl2, 25oC;
[clostripain]: 1.6 µM, [acyl donor]: 2 mM, [H-Ala-Phe-Ala-Ala-Gly-OH]: 4 mM; x = product
yield.
Figure 7
- Partition values of V8 protease-catalyzed peptide coupling using
Z-Pro-Leu-Gly-SCm as acyl donor (32). Conditions: 0.2 M HEPES-buffer, pH 8.0, 37oC;
[acyl donor]: 2 mM, [LAFAKADAFG]: 10 mM, [dipeptide amides, IAAAG]: 20 mM, [enzyme]: 4.9
µM.
Figure 8
- Arrangements of Boc-L-Ala-OGp at the active sites of chymotrypsin (A) and
trypsin (B) derived from the lowest-energy complexes (Günther R, Thust S, Hofmann H-J and
Bordusa F, unpublished results). Shown are the amino acid residues of the enzymes which
either form hydrogen bonds (Asp189/Ser189, Ser190, Ser217/Gly219) or have catalytical
functions (His57, Asp102, and Ser195).
Figure 9
- Yields of chymotrypsin-catalyzed peptide synthesis using 4-guanidinophenyl
esters bearing nonspecific coded and non-coded acyl moieties (Günther R, Thust S, Hofmann
H-J and Bordusa F, unpublished results). Conditions: 0.2 M HEPES-buffer, pH 8.0, 0.2 M
NaCl, 20 mM CaCl2, 25oC, 10% MeOH; [acyl donor]: 2 mM, [acyl
acceptor]: 20 mM, [enzyme]: 0.1-37 µM; all errors are less than 5%.
Figure 10
- General course of peptide ester synthesis via oxime resin strategy (A) and
substrate mimetic-mediated peptide fragment condensation by protease catalysis (B).
Figure 11
- Combination of solid-phase peptide synthesis via oxime resin strategy with
trypsin- (A), V8 protease- (B), and chymotrypsin-catalyzed fragment condensation (C) (38).
Figure 12
- Course of the clostripain-catalyzed coupling of Bz-ß-Ala-OGp and Pbu-OGp
with H-Ala-Phe-Ala-Ala-Gly-OH (44). A, Bz-ß-Ala-OGp; B, Pbu-OGp. Triangles,
Bz-ß-Ala-OGp/Pbu-OGp; squares,
Bz-ß-Ala-Ala-Phe-Ala-Ala-Gly-OH/Pbu-Ala-Phe-Ala-Ala-Gly-OH; circles, Bz-ß-Ala-OH/
Pbu-OH. Conditions: 0.2 M HEPES-buffer, pH 8.0, 0.1 M NaCl, 0.01 M CaCl2, 5%
DMF, 25oC; [acyl donor]: 2 mM, [acyl acceptor]: 4 mM; x = product yield.
Figure 13
- Influence of pentylamine concentration on the product yield x of the
clostripain-catalyzed coupling of Y-OGp with pentylamine (46). Circles, Y = Bz; squares, Y
= Pbu. Conditions: 0.2 M HEPES-buffer, pH 8.0, 0.1 M NaCl, 10 mM CaCl2, 5% DMF,
25oC; [acyl donor]: 2 mM; reaction time: about 15 min.