Carboxypeptidase amino acid sequence

K. R. Cole, S. Kumar, H. L. Trong, R. G. Woodbury, K. A. Walsh, and H. Neurath. Rat mast cell carboxypeptidase amino acid sequence and evidence of enzyme activity within mast cell granules. Biochemistry 50 648-655 (1991). 

An amino acid sequence is ambiguous unless we know the direction m which to read It—left to right or right to left We need to know which end is the N terminus and which IS the C terminus As we saw m the preceding section carboxypeptidase catalyzed hydrolysis cleaves the C terminal ammo acid and so can be used to identify it What about the N terminus ... 

The evolutionary classification has a rational basis, since, to date, the catalytic mechanisms for most peptidases have been established, and the elucidation of their amino acid sequences is progressing rapidly. This classification has the major advantage of fitting well with the catalytic types, but allows no prediction about the types of reaction being catalyzed. For example, some families contain endo- and exopeptidases, e.g., SB-S8, SC-S9 and CA-Cl. Other families exhibit a single type of specificity, e.g., all families in clan MB are endopeptidases, family MC-M14 is almost exclusively composed of carboxypeptidases, and family MF-M17 is composed of aminopeptidases. Furthermore, the same enzyme specificity can sometimes be found in more than one family, e.g., D-Ala-D-Ala carboxypeptidases are found in four different families (SE-S11, SE-S12, SE-S13, and MD-M15). 

Gas-liquid chromatography used for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins was described in 1976 by Davy and Morris. Based on carboxypeptidase A digestion of the protein, the partially digested protein was removed and the amino acids released after known time intervals were analyzed by quantitative gas-liquid chromatography. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins. Thus the amino acid sequences were determined. 

Several enzymes catalyze stepwise removal of amino acids from one or the other end of a peptide chain. Carboxypeptidases232 remove amino acids from the carboxyl-terminal end, while aminopeptidases attack the opposite end. Using chromatographic methods, the amino acids released by these enzymes may be examined at various times and some idea of the sequence of amino acids at the chain ends may be obtained. A dipeptidyl aminopeptidase from bovine spleen cuts dipeptides one at a time from the amino terminus of a chain. These can be converted to volatile trimethylsilyl derivatives and identified by mass spectrometry.233 If the chain is shortened by one residue using the Edman degradation (Section 3) and the dipeptidyl aminopeptidase is again used, a different set of dipeptides that overlaps the first will be obtained and a sequence can be deduced. Carboxypeptidase Y can be used with MALDI mass spectrometry to deduce the C-terminal amino acid sequence for a peptide. However, He and Leu cannot be distinquished. 

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. 

The considerable detail to which we now can understand enzyme catalysis is well illustrated by what is known about the action of carboxypeptidase A. This enzyme (Section 25-7B and Table 25-3) is one of the digestive enzymes of the pancreas that specifically hydrolyze peptide bonds at the C-terminal end. Both the amino-acid sequence and the three-dimensional structure of carboxypeptidase A are known. The enzyme is a single chain of 307 amino-acid residues. The chain has regions where it is associated as an a helix and others where it is associated as a /3-pIeated sheet. The prosthetic group is a zinc ion bound to three specific amino acids and one water molecule near the surface of the molecule. The amino acids bound to zinc are His 69, His 196, and Glu 72 the numbering refers to the position of the amino acid along the chain, with the amino acid at the /V-terminus being number l. The zinc ion is essential for the activity of the enzyme and is implicated, therefore, as part of the active site. 

A. saitoi carboxypeptidase has been used for automatic C-terminal amino acid sequence analyses of anticoagulant decapeptide (SQLQEAPLEK) [83] and a-amylase (-LR) from cultured rice (Oryza sativa) cells [84], and a recombinant Serratia marcesence serine proteinase [85],... 

Figure 18. cDNA sequence of A. saitoi carboxypeptidase and deduced amino acid sequence. 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

In comparison with the marked advancement in sequence determination of the amino terminal region of protein, no good methods have been developed for the determination of the amino acid sequence of the carboxyl terminal region, although several enzymatic and chemical procedures are available. Exoproteinases such as carboxypeptidases A, B, C and Y, have been employed for enzymatic determination. These enzymes catalyze sequential liberation of an amino acid from a carboxyl terminal. However, the reaction efficiency, i.e. velocity and specificity, is not always consistent for all amino acids consisiting of a... 

Traditional methods of C-terminal end determination55 were hydrazine degradation, carboxypeptidase digestion, and tritium labeling. Unfortunately, no chemical methods analyzing amino acid sequence sequentially from the C-terminal end is available with reliability similar to that of Edman degradation. Carboxypeptidase digests a protein and... 

The remaining prohormone contains stretches of amino acids sequences unrelated to biological activity. These sequences are removed by proteolytic enzymes such as endopeptidases which act on dibasic or monobasic amino acid residues, and exopeptidases (e.g., carboxypeptidases and aminopeptidases) which remove amino acid residues from the C-terminal and N-terminal ends, respectively. A single prohormone may yield one or more biologically active and distinct hormones. 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

Figure 2. A) Trypsin hydrolysis/HPLC/thermospray MS analysis of Y-endorphin. The two tryptic fragments (T1 and T ) were separated by HPLC (10% isopropanol in aqueous ammonium acetate (0.1 M) pH 7.1, 1 mL/min) and detected by thermospray MS. B,B ) Thermospray mass spectra of tryptic fragments and Tg separated by HPLC after hydrolysis. C,Cj) Thermospray spectra from trypsin hydrolysis followed by HPLC then carboxypeptidase A, B, and Y hydrolysis for the partial amino acid sequencing of Y-endorphin.

The enkephalins, H—Tyr—Gly—Gly—Phe—X—OH (X=Leu, Met), or so-called opioid peptides because they mimic the action of the opiates, morphine and heroin, have a very short half life in the body because all four peptide bonds are prone to undergoing proteolysis. The Tyr—Gly bond can be hydrolysed by amino-peptidases, the Gly—Gly bond by dipeptidylaminopeptidases, the Gly—Phe bond by enkephalinase and the Phe—Met and Phe—Leu bonds by carboxypeptidases. An enormous number of analogues have been synthesised, especially with the object of producing compounds that exert potent analgaesic action but are free from side effects. Protection of the susceptible bonds by changing the amino-acid sequence is the obvious way to achieve this. The analogue H—Tyr—d—Met—Gly—Phe—... 

The hrst zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by carboxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn " " atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn " "-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule, which is best represented as Zn -OH2. What determines the catalytic properties of each enzyme is not only the nature of the donor ligands, but also the distance that separates them in the amino acid sequence of the protein, lypically (Table 12.1), two of the ligands are separated by only 1—3 amino acids, whereas the third ligand is separated by a longer spacer of between 5 and 196 residues. 

Wintersberger2 isolated from bovine pancreatic carboxypeptidase a peptide containing a thiol group involved in the binding of zinc. He first removed the zinc by exposure to a chelating agent (1,10-phenanthroline) or by denaturation, and then labeled the reactive sulfhydryl group by reaction with DDPM and determined the amino acid sequence. 

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