Specificity determinants of acylaminoacyl-peptide hydrolase.

In an attempt to explore how specific features of the substrate's primary structure may affect the activity of rabbit muscle acylaminoacyl-peptide hydrolase (EC 3.4.19.1), a number of acetylated peptides containing specific amino acid replacements in specific positions were prepared and compared as substrates for the hydrolase. The principal variants were D-Ala, Pro, and positive charges (His, Arg, Lys); in addition, the effect of the length of the peptide was also investigated in a less systematic manner. The substrates were either prepared by direct acetylation of peptides, by extension of the N-terminus with acetylamino acids or acetylpeptides, activated as N-hydroxysuccinimide esters, or by isolation of the N-terminal peptides from naturally occurring acetylated proteins. It was found that D-Ala on either side of the bond to be cleaved (positions 1 and 2) completely inhibited the enzymatic activity, whereas acetylated peptides with D-Ala in positions 3 or 4 were as good substrates as those containing L-Ala. Peptides with Pro in positions 2 were also inactive, and most of the peptides with Pro in the third position were very poor substrates; only the peptide Ac-AAP gave reasonably high activity (30% of Ac-AAA), which was reduced to 1-2% if additional residues were present at the C-terminus (Ac-AAPA, Ac-AAPAA). The presence of a positive charge in positions 2, 3, 4, 5, and 6 gave strong reduction in hydrolase activity varying with the charge's distance from the N-terminus from 0 to 15-20% of the rates obtained with the reference peptides without positive charges.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit muscle extracts catalyze the specific removal of N-acetylmethionine from acetylated peptides.

Rabbit muscle has been found to contain an activity that catalyzes the specific removal of Ac-Met from acetylated peptides. The activity is associated with free ribosomes and microsomes in the rabbit muscle extract but can be removed from these subcellular fractions by exposure to 0.5 M NaCl in the presence of 2 mM MgCl2; only partial removal was achieved with microsomes, but complete removal with ribosomes. A nearly 200-fold enrichment of the activity was achieved by this simple succession of differential centrifugation and salt extraction. Eighteen 14C-acetylpeptides have been tested as substrates for the partially purified activity assaying for the production of free 14C-acetylamino acid by high performance liquid chromatography. None of the peptides containing N-terminal acetylated Ala, Asp, Ser, or Gly were cleaved at a significant rate. Six of a total of eight peptides containing N-terminal Ac-Met were cleaved by the ribosomal extract at different rates. The active substrates varied in length from tri- to undecapeptides. The activity is inhibited by high concentrations of the protease inhibitor phenylmethylsulfonyl fluoride. Based on these observations, we tentatively conclude that the activity satisfy the criteria of a general N-terminal protein processing enzyme: it can remove Ac-Met from most, but not all, N-terminal sequences and appears to be inactive toward the N-terminal acetylamino acids most commonly found in eukaryotic proteins.

The amino acid sequence of Escherichia coli cyanase.

The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

The amino acid sequence of yeast enolase. Preparation and characterization of peptides produced by chemical and enzymatic fragmentation.

Yeast enolase was subjected to chemical and enzymatic fragmentation, and the individual peptides produced were isolated by gel filtration and ion exchange chromatography. The chemical fragmentation was achieved by cleavage at the single cysteine residue with 2-nitro-5-thiocyanobenzoic acid, or at the 5 methionine residues with cyanogen bromide. The assignment of the two 2-nitro-5-thiocyanobenzoic acid fragments to the NH2-terminal or COOH-terminal regions (designated C1 and C2, respectively) of the enolase subunit could be done unequivocally on the basis of NH2-terminal and COOH-terminal analysis, and the same was the case for the NH2-terminal and COOH-terminal cyanogen bromide peptides (designated M1 and M6, respectively). From a comparison of the CNBr peptides from enolase with those from Fragment C1, the identity of methionine peptides M4, of which only the NH2-terminal half is present in C1, and M5, which along with M6 is missing in C1, could also be established. The major enzymatic fragmentation was achieved by tryptic cleavage at the 14 arginine residues after acetylation of the lysine residues. Based on overlaps with methionine peptides, most of the arginine peptides could be ordered in proper sequence during the early phases of the work. Because of the size of several of the primary fragments, secondary cleavages were required for optimal sequencing data. These secondary cleavages were accomplished by digestion with Staphylococcus aureus protease, or by tryptic cleavage at cysteine after aminoethylation.

Posttranslational covalent modification of proteins.

A search for derivatized amino acids in proteins has shown that the extent of posttranslational modification of proteins is quite substantial. While only 20 primary amino acids are specified in the genetic code and are involved as monomer building blocks in the assembly of the polypeptide chain, about 140 amino acids and amino acid derivatives have been identified as constituents of different proteins in different organisms. A brief consideration of the questions about where and when the derivatization reactions occur, how the specificity of the reactions is established, and how the posttranslational modifications can facilitate biological processes, reveal a need for more information on all these points. Answers to these questions should represent significant contributions to our understanding of biochemistry and cell biology.

Effect of glycosylation on the in vivo circulating half-life of ribonuclease.

The circulating half-lives of the four isozymes of bovine pancreatic ribonuclease (RNases A, B, C, and D) have been determined in normal and in nephrectomized rats. The isozymes differ only in their glycosyl content. While A contains no sugars, B has a simple oligosaccharide (GlcNAc2 Man4-5),and C and D each have a complex oligosaccharide (GlcNAc4 Man 2-3 Gal2 Fuc NeuAc2, and GlcNAc4 Man3 Gal2 Fuc NeuAc4, respectively) attached to Asn-34 of the polypeptide chain. All four isozymes were cleared rapidly in normal rats (t 1/2 = 2 to 3 min), as expected on the basis of the established role of the kidneys in removing low molecular weight proteins from circulation. In nephrectomized rats, however, a much slower clearance was observed, thus permitting the evaluation of the role of the carbohydrate chains in the catabolism of the isozymes. The clearance curves can be analyzed in terms of two processes, a rapid initial one, shown to represent the equilibration of the injected enzyme into extravascular space, and a second one which is interpreted as the catabolic clearance of the enzyme. The haf-life of the RNase isozymes was calculated from this second process and found to be in the range 528 to 577 min for RNase A, 15 min for RNase B, 681 to 862 min for RNase C, and 839 to 941 min for RNase D. The rapidly cleared RNase B was treated with alpha-mannosidase to remove 3 of the 4 mannosyl residues, leaving only a trisaccharide (GlcNAc2-betaMan) attached to the protein. The half-life of this RNase B derivatives was found to be in the range 616 to 733 min. From these results it is concluded (a) that the addition of complex oligosaccharides to a protein does not have any significant direct effect on its circulating half-life (RNases C and D compared to RNase A), and (b) that in the rat there exists a mechanism for clearing glycoproteins based on specific recognition of exposed alpha-mannosyl residues (RNase B compared to the other isozymes and to alpha-mannosidase-treated RNase B).