ABSTRACT
The role of each residue of the potato carboxypeptidase inhibitor (PCI) C-terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site-directed mutants: the point substitution of each C-terminal residue by glycine and the sequential deletions of the C-terminal residues. The mutant PCI-CPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined by presteady-state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C-tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI-CPA interaction. Although Tyr37 has a minor contribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C-terminal Gly39 by CPA. The energetic contribution of the PCI secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI-CPA complex.
Subject(s)
Carboxypeptidases/metabolism , Plant Proteins/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/metabolism , Base Sequence , Binding Sites , Carboxypeptidases A , DNA Primers , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Plant Proteins/chemistry , Plant Proteins/genetics , Protease Inhibitors , Protein Binding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
This review briefly surveys various classes of biologically active and flavor peptides that have been isolated and characterized in recent years, and analyzes emerging trends and advances in biotechnological methods for their production.
Subject(s)
Endopeptidases , Enzymes, Immobilized , Oligopeptides/chemical synthesis , Peptides/chemical synthesis , Amino Acid Sequence , Anti-Bacterial Agents/chemical synthesis , Antiviral Agents/chemical synthesis , Biotechnology/methods , Hormones/chemical synthesis , Molecular Sequence Data , Neuropeptides/chemical synthesis , Peptide Fragments/chemical synthesisABSTRACT
The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.
Subject(s)
Neuropeptides/chemical synthesis , Peptide Fragments/chemical synthesis , Amino Acid Sequence , Animals , Insecta , Kinetics , Molecular Sequence Data , StereoisomerismABSTRACT
The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.
