Modification of amino acid residues in the S 1 ′ binding site of carboxypeptidase Y

Svendsen

1984

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Keywords: Carboxypeptidase Y, chemical modification, a m i n o acid sequenceChemical modification studies have previously indicated that the Sj binding site of carboxypeptidase Y contains a cysteinyl residue and that the S] binding site contains a methionyl residue (Carlsberg Res. Commun. 48, 9-19 (1983); 49, 535-554 (1984); 49, 627-638 (1984)). These studies also indicated that the active site contained an additional methionyl residue situated in an as yet unidentified position. In the present paper the positions of these amino acid residues has been identified in the amino acid sequence which has been revised on the basis of the nucleotide sequence (T. STEVENS, personal communications). The cysteinyl residue was identified as Cys-341 after alkylation with J4C-iodoacetic acid followed by separation of the peptides produced by cyanogen bromide cleavage and Edman degradation of the radioactive peptide. The methionyl residue situated in the S; binding site was identified as Met-398 after alkylation with ~4C-iodoacetamide, separation of the peptides produced by cyanogen bromide cleavage and thermal cleavage of the peptide containing the radioactivity. This assignment was confirmed by the failure of cyanogen bromide to cleave at the position ofcarboxypeptidase Y in which this particular methionyl residue had been alkylated or oxidized. Using the same method the other methionyl residue in the active site was identified as Met-313.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of methionyl and cysteinyl residues in the substrate binding site of carboxypeptidase Y

Svendsen

1984

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“…KUHN et al (113) have described that alkylation ofa methionyl residue in carboxypeptiase Y with iodoacetamide causes a reduction in the peptidase activity of the enzyme without significantly altering the esterase activity. In this laboratory, this methionyl residue has been identified as Met-398 in the amino acid sequence (36) and in addition it has been alkylated with phenacylbromide, m-nitrophenacylbromide and p-nitrophenacylbromide (34) and oxidized with H202 (35). All these derivatives of carboxypeptidase Y have been separated from residual unmodified enzyme by affinity chromatography and characterized kinetically (34,35).…”

Section: Modification At the S; Binding Sitementioning

confidence: 99%

“…In this laboratory, this methionyl residue has been identified as Met-398 in the amino acid sequence (36) and in addition it has been alkylated with phenacylbromide, m-nitrophenacylbromide and p-nitrophenacylbromide (34) and oxidized with H202 (35). All these derivatives of carboxypeptidase Y have been separated from residual unmodified enzyme by affinity chromatography and characterized kinetically (34,35). Modification of carboxypeptidase Y with phenacylbromide influences KdK~ for the hydrolysis of ester and amide substrates in a manner which is dependent on the size of the leaving group of the substrate (Table VIII).…”

Section: Modification At the S; Binding Sitementioning

confidence: 99%

Serine carboxypeptidases. A review

1986

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180

130

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...

“…Since the amino acid occupying position 398 is part of the S~ binding site (8,9) Cys-CPD-Y was characterized kineticaUy using a series of FA-ester, FA-peptide and FA-amide substrates with different groups in the P; position. Exchange of Met-398 with a cysteinyl residue resulted in a reduction in k~,dK~ for all the substrates listed in Table I and [I (with the exception of FA-Ala-OBzl) due to decreased k~, values and increased Km values.…”

Section: Peptide Synthesis and Deamidation Of Peptide Amidesmentioning

confidence: 99%

Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis

Bech

1988

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It is demonstrated that site-directed mutagenesis successfully can be combined with chemical modification creating enzyme derivatives with altered properties. A methionyl residue located in the S] binding site of carboxypeptidase Y was replaced by a cysteinyl residue and the mutant enzyme was isolated and modified with various alkylating and thioalkylating reagents. Treatment of the mutant carboxypeptidase Y with bulky reagents like phenacyl bromide and benzyl methanethiolsulfonate caused a drastic reduction in the activity towards substrates with bulky leaving groups in the P] position, i.e. -OBzl, -VaI-NH2 and amino acids (except -Gly-OH), while substrates with small groups in that position, i.e. -OMe and -NH2, were hydrolysed with increased rates. The presence of a positive charge, in addition to a bulky group, had a further adverse effect on the activity towards substrates with large leaving groups, whereas the activity towards those with small leafing groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsuifonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively. SCHECHTER and BERGER (17). Accordingly, the binding site for the C-terminal amino acid residue is denoted S~ and those for the amino acid residues in the amino-terminal direction away from the scissile bond are denoted S~,S2,...,S,. Amino acids in the substrate are referred to as Pt,P2,-..,P. and P~ in correspondence with the binding sites. Springer-Verlag