Influence of guanidine derivatives on the specificity of malt carboxypeptidase

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Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes.Most serine carboxypeptidases presumably participate in the intracellular turnover of proteins and some of them, in addition, release amino acids from extracellular proteins and peptides. However, prolyl carboxypeptidase which cleaves the C-terminal peptide bond of angiotensin II and III is a serine carboxypeptidase with a more specific function.Serine carboxypeptidases are usually glycoproteins with subunit molecular weights of 40,000 -75,000. Those isolated from fungi apparently contain only a single peptide chain while those isolated from higher plants and animals in most cases contain two peptide chains linked by disulfide bridges. However, a number of the enzymes aggregate forming dimers and oligomers.It is probable that the well-known catalytic mechanism of the serine endopeptidases is also employed by the serine carboxypeptidases but presumably with the difference that the plQ of the catalytically essential histidyl residue is somewhat lower in the carboxypeptidases than in the endopeptidases. However, the leaving group specificity of these two groups of enzymes differ since the carboxypeptidases only release C-terminal amino acids from peptides (peptidase activity) and not longer peptide fragments. In addition, they release C-terminal amino acid amides (peptidyl amino acid amide hydrolase activity) or ammonia (amidase activity) from peptide amides and alcohols from peptide esters (esterase activity) and this property they share with the serine endopeptidases. Like other proteolytic enzymes the serine carboxypeptidases contain binding sites which secure the interaction between enzyme and substrate. In this laboratory, the properties of these have been studied for three serine carboxypeptidases, i.e. carboxypeptidase Y from yeast and malt carboxypeptidases I and II, by means of kinetic studies, chemical modifications of amino acid side-chains located at these binding sites and exchange of such amino acid residues by site-directed mutagenesis.Serine carboxypeptidases, such as carboxypeptidase Y and malt carboxypeptidase II which are available in large quantities, can be applied for several purposes. Their broad specificity and ability to release amino acids from the C-terminus of a peptide chain can be employed in determination of amino acid sequences, and their ability to catalyze transpeptidation reactions and aminolysis ofpeptide esters can be employed to exchange C-terminal amino acid residues in peptides and in step-wise synthesis ofpolypeptides, respectively. The type of reactions catalyzed by these enzymes is l...

Section: E + S E Es -) Es' > E + P2supporting

confidence: 61%

Section: Ji111111111111~]1]]]1]]]]]1~111111///111111]]]//]]1~mentioning

confidence: 56%

Section: Studies With Modifiersmentioning

confidence: 75%

Section: Ji111111111111~]1]]]1]]]]]1~111111///111111]]]//]]1~mentioning

confidence: 96%

Section: The Influence Of the Substrate Structurementioning

confidence: 99%

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Serine carboxypeptidases. A review

Breddam

1986

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180

130

Malt carboxypeptidase catalyzed aminolysis reactions

Breddam¹,

Ottesen²

1984

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Malt carboxypeptidase catalyzes the formation of peptide bonds using N-benzoyl-arginine esters as acyl components and amino acid amides or amino acid methyl esters as nucleophiles. With seven different nucleophiles yields of 50-95% were obtained while no aminolysis was observed with H-Pro-NH2 and H-GIu-a-NH2. The enzyme is easily saturated with nucleophile indicating the formation of a complex between nucleophile and acyl-enzyme intermediate prior to deacylation. The nature of the side-chain of the amino acid esters used as nucleophiles strongly influences the apparent dissociation constant of this complex (KN~app~) while less dependence on the side-chain is observed with amino acid amides. KN~app~ is drastically increased by the presence ofphenylguanidine in the reaction medium suggesting a competition between this substance and the nucleophile for the same binding site. This site has previously been identified as the S~ binding site of malt carboxypeptidase (Carlsberg Res. Commun. 48,573-582 (1983)). The influence of the nucleophile H-VaI-NH2 on the kinetic parameters for the cleavage of N,-CBZ-Lys-p-nitrophenylesler indicates that the acylation step is rate-limiting, and that H-Val-NH2 has two different modes of binding to the enzyme.

Carboxypeptidase S-1 from Penicillium janthinellum: Enzymatic properties in hydrolysis and aminolysis reactions

Breddam

1988

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Carboxypeptidase S-1 from Pencillium janthinellum has been isolated by affinity chromatography and characterized. The enzyme activity is unusually stable in organic solvents, e.g. 80% methanol. The hydrolysis of peptide substrates is apparently dependent on three ionizable groups. One group, with pKa of 4.0-4.5, is a catalytically essential residue in its deprotonated form, and another group with a pKa of 6.5-7.0 functions in its protonated form, apparently as the binding site for the C-terminal carboxylate group of peptide substrates. The third group, with a pKa of 5.0-5.5, appears to be a carboxylic acid group located at the S~ binding site. Deprotonation of this group to form a negatively charged carboxylate group has an adverse effect on the hydrolysis of substrates with hydrophobic amino acid residues at the P~ position and a beneficial effect on the hydrolysis of substrates with the positively charged arginyl or lysyl residues at this position. The substrate preference of the enzyme is consequently pH dependent. At pH 6.0 (the optimum for ester hydrolysis) the enzyme is essentially specific for Bz-X-OMe substrates where X = Arg and Lys.Using amino acids and amino acid amides as nucleophiles carboxypeptidase S-I efficiently catalyses the formation of peptide bonds by aminolysis of peptides (transpeptidation reactions) and peptide esters provided that the substrate contains a basic amino acid residue at the Pt position, e.g. Bz-Arg-OBu and Bz-Arg-Leu-OH. With several nucleophiles the fractions of aminolysis exceed those previously reported in similar studies with carboxypeptidase Y and malt carboxypeptidase II.Abbreviations: Bicine = N,N-bis(hydroxyethyl)glycine; Bu = butyl; Bz = N-benzoyl; CPD-M~, CPD-MH and CPD-MH~ = malt carboxypeptidases I, II and III, respectively; CPD-Y = carboxypeptidase Y; CPD-S-I = penicillocarboxypeptidase S-1; EDTA = ethylene diamine tetraacetic acid; FA = furylacryloyl; Hepes = N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid; HPLC = high performance liquid chromatography; Mes = 2-(N-morpholino)ethane sulfonic acid; Z = N-carbobenoxy. Other abbreviations of amino acids, amino acid derivatives and peptides are according to the guidelines of the IUPAC-IUB Commission on Biochemical Nomenclature. The binding site notations for the enzymes is that of SCHECHTER and BERGER (20). Accordingly, the binding site for the C-terminal amino acids residue of the substrate is denoted S~' and those for the amino acid residues in the amino-terminal direction away form the scissile bond are denoted S~, Sz..., S,. The substrate positions are all denoted Pt', Pt, P2...Po in correspondence with the binding sites. Springer-Verlag