Biomedical subjects

C Groeger

Publications and source records attributed to C Groeger.

4 recordsLinked to original sources

PubMed1994

BPTI backbone variants and implications for inhibitory activity.

Structural variants of BPTI were synthesized en route an enzymatic-chemical semisynthesis. The P1-P2 amide bond of the inhibitor molecule, which, as donor, contributes a hydrogen bond towards trypsin in the enzyme-inhibitor complex, was replaced by either a ketomethylene function or an ester bond yielding molecules with inhibitory activity. The two backbone-mutated BPTI derivatives showed increased dissociation constants of their respective trypsin complexes, obviously due to the lack of a single hydrogen-bond interaction in the enzyme-inhibitor complex.

Aprotinin↗

PubMed1991

Enzymatic semisynthesis of aprotinin homologues mutated in P' positions.

The replacement of amino acids in the P'1 and P'2 position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the "modified" inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys15-Ala16, the residues P'1 (Ala16) and P'2 (Arg17) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a "one pot" reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala16-Arg17 was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg17-Ile18 peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys15 and Xaa16 are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P'1 residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.

Amino Acid Sequence↗

PubMed1991

Chemical semisynthesis of aprotinin homologues and derivatives mutated in P' positions.

An extended concept for the replacement of amino acids in the P' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys15-carboxyl group) lacking the amino acids Ala16 and Arg17 leads to aprotinin homologues and derivatives mutated in the P'1 and P'2 position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala17]BPTI and [seco-17/18]BPTI is described in detail.

Amino Acid Sequence↗

PubMed1991

Enzymatic fragment substitution as a tool in protein design.

An easy and rapid enzymatic method is described which allows replacement of P'-residues in bovine pancreatic trypsin inhibitor. Insertion of Xaa-Arg or Xaa-Lys into a BPTI fragment lacking P1' = Ala16 and P2' = Arg17 was carried out in a "one pot" reaction catalysed by trypsin in the presence of 80% 1,4 butanediol.

Amino Acid Sequence↗