Yeast carboxypeptidase

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Knop M, Hauser N, Wolf DH (1996a) N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast. Yeast 12 1229-1238... 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

Comparison of the deduced sequence of A. saitoi carboxypeptidase with other known serine carboxypeptidase sequences shows that they share a low degree of similarity 32% with wheat carboxypeptidase II, 32.3% with malt carboxypeptidase II and 26.2% with yeast carboxypeptidase Y (Figure 19) [88], However, all of the sequences conserve the catalytic domains (indicated by boxes II to IV in Figure 19) and the domain (box I in the Figure 19) which contains the amino acid residues recognizing the C-terminal carboxylate group of peptide substrates. There are also present in the sequence ten potential sites for N-linked glycosylation. 

The A. saitoi carboxypeptidase cDNA was cloned downstream of a GDP promoter, and the resulting plasmid, pGCP13, was used to generate recombinant A. saitoi carboxypeptidase protein. No enzymatic activity was detected in the culture supernatant. We detected the A. saitoi carboxypeptidase activity of the extract obtained from yeast cells transfected with A. saitoi carboxypeptidase cDNA in forward orientation (pGCP13), although no activity was observed with the vector alone at pH 3.1. The recombinant A. saitoi carboxypeptidase activity... 

Western-blot analysis of yeast cell extracts shows a 72 kDa protein with rabbit anti-(A saitoi carboxypeptidase) serum, which is consistent with the apparent molecular mass of the native A. saitoi carboxypeptidase. Conversely, the extracts obtained from yeast cells transfected with the vector (pG-3) alone or with cDNA in reverse orientation (pGP31), as negative controls, yielded no stainable protein. 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

Two recently isolated serine proteases have quite different specificities. One is a protease of Staphylococcus aureus which has a high specificity for glutamic acid residues at Pi 16, 17). The other is the yeast carboxypeptidase listed in Table I 11), As the name indicates, it degrades polypeptides by cleaving amino acid residues from the C-terminal end of the chain—a most unexpected specificity for a serine protease. Unlike Carboxypeptidase A, it is stable, is capable of removing proline residues, and would seem to be an ideal enzyme for determining C-terminal sequences. 

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

Analyses of in situ DNA synthesis of Euglena gracilis identify zinc-dependent steps in the eukaryotic cell cycle and show that the derangements in RNA metabolism are critical determinants of the growth arrest associated with zinc deficiency. Combined use of microwave-induced emission spectrometry and micro gel emulsion chromatography shows the presence of stoichiometric amounts of zinc essential to the function of E. gracilis and yeast RNA polymerases, the reverse transcriptases" from avian myeloblastosis, murine leukemic and woolly type C viruses, and E. coli methionyl tRNA synthetase. These results stress the importance of zinc to both nucleic acid and protein metabolism. Transient-state kinetic studies of carboxypeptidase A show that zinc functions in the catalytic step of peptide hydrolysis and in the binding step of ester hydrolysis. 

Higher concentrations of aa -D are required to bring about 50 % inhibition than are necessary for OP, as is the case for carboxypeptidase. This is presently explained on the basis of an additional degree of freedom in the aa -D molecule as compared with OP, owing to rotation about the C—C bond, resulting in a lower statistical probability for chelation with aa -D. The degree of inhibition may be related to the stability of the zinc complexes formed, but while consistent with this parameter, other factors enter which have been discussed above for carboxypeptidase (see p. 352, also Table VIII) and which apply for yeast ADH. 

Figure 3.4 Catalytic mechanism of yeast carboxypeptidase Y mediated peptide hyrolysis and a model for the acyltransferase activity of SMT.

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