Peptide substitution kinetics

Substitution Kinetics of Copper(II)—Peptide Complexes. Three main reaction pathways have been found for the displacement of copper from peptide complexes—(1) proton transfer to the peptide group, (2) nucleo-... 

Iridium can be removed from Cu(III)—peptide solutions by passing them through anion exchange columns. The resulting Cuin(H 3G4) is much slower to decompose in acid than is the Cun(H 3G4)2" complex. In neutral solutions at 25° the half-life of Cum(H 3G4) is about 1 hr. The decomposition rate increases in base as well as in acid. The substitution kinetics of the Cu(III) (ds) complexes are clearly much slower than the corresponding Cu(II) complexes. This fact was used in choosing Chelex ion exchange resin to remove Cu(II) from Cu(III) in order to determine the molar absorptivity of Cum(H 3G4). This value of 7200 d= 300 M"1 cm"1 at 365 nm was checked by several other methods (9). 

Choulier, L., Rauffer-Bruyere, N., Ben Khalifa, M., Martin, F., Vernet, T. andAltschuh, D. (1999), Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent... 

The kinetics and mechanisms of substitution reactions of metal complexes are discussed with emphasis on factors affecting the reactions of chelates and multidentate ligands. Evidence for associative mechanisms is reviewed. The substitution behavior of copper(III) and nickel(III) complexes is presented. Factors affecting the formation and dissociation rates of chelates are considered along with proton-transfer and nucleophilic substitution reactions of metal peptide complexes. The rate constants for the replacement of tripeptides from copper(II) by triethylene-... 

The individual contributions of the H20, H+, and HO- catalysts to the mechanism of the reaction were further evaluated by means of the kinetics parameters (Table 6.4). At neutral pH, Reactions a and c were both dominated by fcH2<> The second-order rate constants ku+ and kHO- were identical, indicating similar efficiencies of the H+ and HO catalysts. Interestingly, the second-order rate constants for the hydrolysis of Gly-D-Val (6.48) to yield Gly and D-Val (6.49) (Reaction b) could also be calculated (Table 6.4). The similarity to the corresponding rate constants of Reactions a and c suggests that the rate of peptide bond hydrolysis is not particularly sensitive to substitution at or protonation of the flanking amino and carboxy groups [69],... 

Outside Protonation. When metal-peptide complexes are placed in acidic solutions, the complexes dissociate. Metal ions which are sluggish in their substitution reactions, such as Ni(II) (43), Pd (II) (44), and Co (III) (45), add protons to the peptide oxygens prior to the metal-N (peptide) bond dissociation. The Cun(H 2GGhis)" complex is sufficiently sluggish in its reaction with acid to permit outside protonation to be observed kinetically (12). Protonation constants of 104 2 and... 

The application of asymmetric peptides is much less frequently described (Cai et al., 2001). The production of asymmetric RhllO-labeled to-substituted peptides is much more difficult because the synthesis requires significantly more steps than required for symmetrically to-substituted peptides. The critical step is the synthesis of the mono-substituted intermediate product RhllO-ZYX with low yield in most cases (personal communication). However, due to the fact that a strong fluorescence increase is only observed after the fluorophore has been cleaved off from both peptide chains of the symmetrically to-substituted substrate (two-step proteolysis), the kinetics of the enzymatic reaction can be followed more accurately and easily with peptides comprising only one copy of the scissile bond. 

The water molecules are substitutionally labile and the soft M(I)(C0)3+ species reacts with ligands containing both hard and soft ligands, for example, thioethers, histidine, and carboxylates, to form kinetically stable octahedral complexes. In fact, histidine in a peptide is such a strong binding ligand that it often competes with the intended bifunctional ligand. 

Even though this dipeptide is turned over quite slowly, the complex examined is probably a non-productive one. Furthermore an analogous ester substrate has not been found, and it is known that carboxypeptidase behaves quite differently toward ester and peptide substrates. In particular, the kinetic parameters for peptide hydrolysis for a series of metal substituted carboxypeptidases indicate that fccat values can range from 6000 min for the cobalt enzyme down to 43 min for the cadmium enzyme 66). The values on the other hand are almost totally independent of the particular metal present. The exact opposite is true for ester hydrolysis. Km varies from 3300 M for the cobalt enzyme to 120 M for the cadmium enzyme while k<.at is essentially unchanged. 

Fig. 3. Quantification of Bcl-2 family activities with the long-format cytochrome c release assay. Results are the average of duplicate measurements in (A) and are averages of triplicate measurements +/-SEM in (B-D) (many error bars in panels [B-D] are obscured by the symbols). In panels (A-D) open circles indicate cytochrome c detected when Triton X-100 was added to mitochondria and open diamonds indicate cytochrome c detected when no Bcl-2 family proteins were added to mitochondria. All incubations of mitochondria except those in (B) were for 30 min. (A) Recombinant human Bid (solid circles) and caspase-8-cleaved human Bid (squares) induce release of cytochrome c from isolated mouse liver mitochondria in a dose- dependent manner. (B) Kinetics of 52 nM (solid squares) and 5.2 nM (solid circles) human cleaved Bid-induced cytochrome c release from isolated mouse liver mitochondria. (C) Bcl-xL inhibition of caspase-8-cleaved human Bid and cleaved mouse Bid induced cytochrome c release. Mitochondria were incubated with 52 nM cleaved human Bid without (solid diamond) or with ( solid squares) the indicated concentrations of mouse Bcl-xL. Mitochondria were also incubated with 17 nM cleaved mouse Bid without (open square) or with (solid circles) the indicated concentrations of mouse Bcl-xL. (D) A synthetic peptide (GQVGRQLAIIGDDINR) corresponding to the amino acid 72-87 BH3 region of Bak prevents Bcl-xL from inhibiting caspase-8-cleaved mouse Bid induction of cytochrome c release. Mitochondria were incubated with 17 nM caspase-8-cleaved mouse Bid without (triangle) or with (open square) 155 nM mouse Bcl-xL and the indicated concentrations of Bak-BH3 (solid circle) or the corresponding Bak peptide (GQVGRQAAIIGDDINR) with a L to A substitution (solid squares).

Colman and Frieden (108) demonstrated in 1966 that acetylation of one amino group per subunit with acetic anhydride produces 80% inactivation. More extensive acetylation alters the degree of polymerization and certain kinetic parameters (276). Almost simultaneously, Anderson et al. (277) reported the reversible inhibition of GDH by pyridoxal 5 -phosphate and certain other aromatic aldehydes. The inhibition was attributed to formation of a Schilf base since reduction with NaBH4 results in irreversible inactivation. It was estimated that approximately one residue of -pyridoxyllysine had been formed per subunit. In 1969, Holbrook and Jeckel (278) inactivated the enzyme by reaction with a substituted maleimide and, subsequently, obtained the partial sequence of a tryptic peptide containing a modified lysine residue (Fig. 7). 

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