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

Abstract

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

A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the α/β hydrolase fold, acts as a pH‐dependent protein‐stabilizing element

“…The second part of the hypothesis predicts an adverse effect on stability of the 2 glutamic acids when they both are ionized due to charge repulsion, consistent with previous observations that serine carboxypeptidases generally are rather unstable at basic pH (Breddam et al, 1983Breddam, 1988). Unfortunately, the pK, of the second deprotonation cannot be determined by kinetic investigations, and this prevents studying the stability at a pH where a defined proportion of the enzyme is in the double-deprotonated form.…”

Cloning, Expression, and Substrate Specificity of MeCPA, a Zinc Carboxypeptidase That Is Secreted into Infected Tissues by the Fungal Entomopathogen Metarhizium anisopliae

“…and nitrogen source derepressed CPA secretion, but the presence of host cuticle or proteins was required for secretion of high levels of MeCPA. Secretion was repressed when NH 4 Cl was added to media containing protein showing that nitrogen repression overrides the enhancing effect of polymeric substrates. Consistent with regulation at the level of transcription, a 1.2-kilobase MeCPA transcript is present in very low abundance in a peptone medium but is highly expressed during growth on insect cuticle (Fig.…”

“…These are traditionally classified into the A types (with a preference for apolar COOH-terminal residues) and the B types (with a preference for basic COOH-terminal residues) (1,2). In contrast, actinomycetes (bacteria) produce carboxypeptidases with a dual A ϩ B specificity toward both neutral and basic substrates and the distinct vertebrate A and B types presumably arose from such a precursor (3,4). Why or when in the course of evolution a single carboxypeptidase with both A and B specificities was abandoned in favor of multiple enzymes with more limited specificities is not known.…”

Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic ArchaeonThermococcussp. NA1

“…Many carboxypeptidases exhibiting broad substrate specificities have been reported. 22) On the basis of preferences for amino acids at the C-terminus, Penicillium janthinellum CP, 23) a member of the serine carboxypeptidases, and carboxypeptidase T from Thermoactinomyces sp. 24) cleaved the peptide bonds formed by basic and hydrophobic Cterminal amino acid residues, whereas carboxypeptidase A and carboxypeptidase C, and carboxypeptidase B and carboxypeptidase D exhibited strong preferences for hydrophobic and basic residues respectively.…”