Jack T. Johansen

Structural properties of the zinc site in Cu,Zn-superoxide dismutase; Perturbed angular correlation of gamma ray spectroscopy on the Cu, 111Cd-superoxide dismutase derivative

Abstract The Zn(II) site of the dimeric Cu(II),Zn(II)-superoxide dismutase from Saccharomyces cerevisiae has been examined by means of perturbed angular correlation of gamma rays (PAC) on the Cu(II),Cd(II)- and Cu(I),Cd(II)-superoxide dismutase. The PAC spectrum for the Cu(II),Cd(II) enzyme reveals two different, pH independent, coordination geometries for the Cd site. Removal of Cu(II) does not affect the PAC spectrum, which suggests that Cu(II) and Cd(II) do not share a common histidine side chain as ligand. The results are consistent with either an equilibrium between two coordination geometries for Cd(II) in each subunit or a difference in the structure of the Cd(II) site in the two subunits. In contrast, in the reduced enzyme only one structure is present, identical for the two subunits.

Isolation of carboxypeptidase Y by affinity chromatography

Carboxypeptidase Y from bakers’ yeast has been purified in high yields by affinity chromatography. The affinity gel was prepared by coupling the specific inhibitor p-aminobenzylsuccinic acid via an azo linkage to Sepharoseglycyl-tyrosine. This affinity gel was able to bind carboxypeptidase Y specifically and quantitatively from a crude yeast autolysate.The isolated enzyme appeared homogeneous by gel electrophoresis and ultracentrifugation, while isoelectric focusing revealed the presence of two components with isoelectric points of pH 3.56 and 3.66, respectively. Small differences in amino acid composition and enzymatic properties between the enzyme from danish yeast and the corresponding enzyme isolated from Fleichmann yeast suggested the existence of more than one form of this enzyme.

The complete amino acid sequence of copper, zinc superoxide dismutase from Saccharomyces cerevisiae

The amino acid sequence of the copper zinc superoxide dismutase from Saccharomyces cerevisiae has been determined by automated Edman degradation. Peptides were obtained from cyanogen bromide cleavage, Staphylococcus aureus V8 protease digestion, tryptic and chymotryptic digests of the citraconylated reduced and carboxymethylated enzyme, and by further fragmentation of selected peptides with trypsin. From the alignment of these peptides and the previously published sequence of the first 54 amino terminal residues (24) the complete sequence was deduced by direct sequence identification of all 153 amino acid residues and of all peptide overlaps. The amino acid sequence corresponds to a molecular weight of 15,950 for each of the two identical subunits in the native enzyme. The primary structure of yeast copper, zinc superoxide dismutase is 55% identical with the sequence of the copper, zinc enzyme from bovine erythrocytes. Importantly, all the copper and zinc ligands, six histidine residues and one aspartate residue from the bovine enzyme, are conserved in the yeast enzyme. The high overall sequence homology and conservation of important metal binding active site amino acid residues suggest that the three-dimensional structure and in particular the active site geometry is virtually the same for the bovine and yeast enzyme. In contrast no sequence homology is apparent by comparison with the manganese or iron class of superoxide dismutases indicating that the two classes have not evolved from a common ancestor.

ENZYMATIC PEPTIDE SYNTHESIS. CARBOXYPEPTIDASE Y CATALYZED FORMATION OF PEPTIDE BONDS

It is demonstrated that carboxypeptidase Y from Saccharomyces cerevisiae can catalyze the formation of peptide bonds using N-acylamino acid esters as substrates and free amino acids or amino acid amides as nucleophiles. The coupling yields observed with free amino acids were max. 60% for alanine and lysine and they depended strongly on the reaction parameters; viz. pH, temperature and concentration of the amino acids as well as their structures. Importantly, under the conditions of peptide synthesis, the peptide product is not hydrolyzed. Amino acid amides were incorporated in higher yields (60–95%) which were less sensitive to the experimental conditions and the structures of this type of nucleophile. The present study suggests that carboxypeptidase Y, having a broad specificity for amino acid side chains, may become a general catalyst for enzymatic peptide synthesis in the homogeneous phase.

Carboxypeptidase Y catalyzed transpeptidations and enzymatic peptide synthesis

It is shown that carboxypeptidase Y, in addition to its known amidase and peptidase activities, also exhibits peptidyl-amino-acid-amide hydrolase activity, i.e., amino acid amides are released from peptide amides. These three activities were investigated as to their usefulness in catalyzing transpeptidation reactions in the presence of suitable nucleophiles. Using peptides and peptide amides as substrates and amino acids or amino acid amides as nucleophiles, it could be shown that transpeptidation products were formed in accordance to each of the above mentioned activities of carboxypeptidase Y. While transpeptidation via the amidase activity results in an elongation of the peptide chain, transpeptidation via peptidase activity results in the exchange of the C-terminal amino acid residue. If the substrate is a peptide amide, transpeptidation via the peptidyl-amino-acid-amide hydrolase activity will result in the formation of a peptide where the C-terminal amino acid amide is replaced by either an amino acid or an amino acid amide.As conditions have not yet been found where all three activities are well separated from each other, it is not generally possible to direct these transpeptidations towards a single desired product in high yield. However, the data presented in this paper indicate that for peptides of certain unique amino acid sequences the carboxypeptidase Y catalyzed transpeptidations may be applicable in the field of enzymatic peptide synthesis.

Carboxypeptidase Y catalyzed peptide synthesis using amino acid alkyl esters as amine components

Carboxypeptidase Y catalyzed transacylation reactions between N-protected amino acid methyl esters or peptide methyl esters as initial acyl components and methyl, ethyl, isopropyl or tert.-butyl esters of different α-amino acids as amine components are described. The yield of peptide bond formation and the extent of oligomerization of the amine components both depend on the nature of their side chain as well as on the nature of their alkyl ester group. Thus, the methyl esters of all hydrophobic amino acids that were tested oligomerized to various extents (in total product yields ranging from 60–90%). On the other hand, the methyl esters of the hydrophilic amino acids did not oligomerize, and were incorporated in yields ranging from 25–50 %. Increasing the size of the ester group from methyl, to ethyl, to propyl etc. resulted in a drastic decrease of the degree of oligomerization and — with the exception of glycine — led to reduced coupling yields.

Influence of the structure of amine components on carboxypeptidase Y catalyzed amide bond formation

Carboxypeptidase Y catalyzed reactions between a common acyl component (Bz-Ala-OMe) and a variety of amine compounds are described. The α-amino acid amides tested were—with the exception of isoglutamine— incorporated in high yields (70–95%). For the free α-amino acids, the yields were below 60% and fluctuated widely. For the cases examined, the enzyme only reacted with thel-enatiomers of these compounds. Dipeptides were not accepted as amine components. Glycinonitril, glycine-n-methyl amide and threonine-n-methyl amide reacted in low yields (<20%) only. None of the secondary amines tested (sarcosine, sarcosine methyl ester,n-methyl-alanine, proline and proline amide) was incorporated to form an amide bond. However, several structurally interesting primary amines (e.g.,l-alaninol, cyclopropylamine, β-alanine amide, taurine etc.) reacted in good yields (40–60%), indicating that carboxypeptidase Y may also become useful for the synthesis of certain peptide analogues.

Metal coordination geometry and mode of action of carbonic anhydrase. Effect of imidazole on the spectral properties of Co(II) and111Cd(II) human carbonic anhydrase B

The effect of the inhibitor imidazole on the visible absorption spectrum of Co(II) human carbonic anhydrase B and on the perturbed angular correlation spectrum of111Cd(II) human carbonic anhydrase B have been studied at various pH values. It is demonstrated that imidazole binds both to the111Cd(II) and Co(II) enzymes and perturbs their respective spectra. Furthermore, spectral titrations of Co(II) with imidazole reveals binding constants similar to those determined kinetically for both the Zn(II) and the Cd(II) enzymes. Imidazole binds to both the low and high pH forms of the enzyme, and have only a weak effect on the activity linked ionization. The perturbed angular correlation results suggest that the human carbonic anhydrase B imidazole complex is five coordinated with a hydrolytic water molecule, imidazole and from the protein part His-94, His-96 and His-119 as the ligands.The effect of acetazolamide binding of the perturbed angular correlation spectrum suggest, that this inhibitor also forms a five coordinated complex in which the NH− and an oxygen atom of the sulfonamide group displace one or two water molecules.Detailed analysis of the perturbed angular correlation data for111Cd(II) human carbonic anhydrase B suggests that the low pH form of the enzyme is four coordinated with a water molecule as the fourth ligand, whereas the high pH and active form of the enzyme might be five coordinated with a hydroxyl ion and a water molecule as the fourth and fifth ligands, respectively.A mechanism for CO2 hydration is proposed in which the metal is five coordinated and functions both as aLewis acid, activating CO2 through direct coordination, and by activation of a coordinated water molecule for nucleophilic attack on CO2.

CARBOXYPEPTIDASE Y CATALYZED C-TERMINAL MODIFICATIONS OF PEPTIDES

It is demonstrated that carboxypeptidase Y catalyzes the exchange of C-terminal amino acid residues in peptides for various other groups. Using N-blocked dipeptides as substrates and alcohols, ammonia, glycine, glycine amide and glycine methyl ester as nucleophiles, it is shown that peptides can be converted to peptide esters, peptide amides and to peptides with altered C-terminal amino acid residues. The incorporation of the different amine nucleophiles could be studied in a wide pH range, since the products were resistant towards further degradation. However, the conversion of peptides to peptide esters by alcoholysis was limited to pH below 4, due to enzymatic hydrolysis of the reaction product. The pH profile for the incorporation of the various amine nucleophiles suggests that they bind at the same location as the C-terminal amino acid residue of the peptides. The binding of glycine amide and glycine methyl ester to the enzyme, prior to aminolysis, is dependent on the state of ionization of a residue with a pKa of 6.6–7.1.It is further demonstrated that the coupling yields for all the transacylation reactions, catalyzed by carboxypeptidase y are dependent on the hydrophobicity of the amino acid leaving the active site. A pronounced influence of the penultimate residue of the substrate is demonstrated as well. The implications are discussed.

The complete amino acid sequence of manganese-superoxide dismutase from Saccharomyces cerevisiae

The complete amino acid sequence of manganese superoxide dismutase isolated from Saccharomyces cerevisiae has been determined by automated Edman degradation. Peptides for sequence analysis were produced by cleavage with cyanogen bromide, hydroxylamine and S. aureus protease V8. The native enzyme consists of four identical polypeptide chains 203 residues long each containing a single sulfhydryl group and no disulfide bridges. A subunit molecular weight of 22,690 was calculated from the complete sequence. The sequence exhibits a significant degree of homology with the managanese superoxide dismutase from E. coli and B. stearothermophilus.