Biomedical subjects
Publications and source records attributed to K Breddam.
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In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme. To avoid this additional experimental effort, we have developed a method utilizing reversed-flow affinity elution. The method has been successfully employed to purify hundreds of carboxypeptidase Y mutant enzymes.
The high activity of carboxypeptidase S1 with substrates having basic P1 residues is predicted to depend on the size of residue 312 in combination with the presence of a counter-charge in an alpha-helix above the S1 binding pocket. This hypothesis is tested by the construction of 32 mutant forms of carboxypeptidase Y that combines a reduction in size of residue 312 and the introduction of either a basic or an acidic residue at either position 241 or position 245. Kinetic characterization using substrates with Leu, Arg, Lys, Glu, or Asp in P1 demonstrates that most of these enzymes exhibit drastically altered catalytic properties. One mutant enzyme, N241D + W312L, hydrolyzes FA-Arg-Ala-OH with a kcat/KM value of 13 000 min-1 mM-1 corresponding to a 930-fold increase relative to the wild-type enzyme. This increased activity is due to an increase in kcat and is independent of ionic strength. The pH profile of kcat/KM exhibits an optimum around pH 5.5 similar to that observed for CPD-S1. Another mutant enzyme, L245R + W312S, hydrolyzes FA-Glu-Ala-OH and FA-Asp-Ala-OH with kcat/KM values of 5100 and 5300 min-1 mM-1, respectively, corresponding to 120 and 170-fold increases relative to wild-type values. With the latter substrate, a 280-fold reduction of KM is observed. The activity of L245R + W312S is also independent of ionic strength and displays a virtually unaltered dependence on pH. The P1 substrate preference of these two mutant enzymes for Arg versus Asp differs 2.5 x 10(6)-fold. values of single and double mutants demonstrate that the effects of reducing the size of Trp312 and introducing a charged residue at position 241 or 245 in some cases exceed 100% additivity. Thus, the double mutant enzyme gains more activation energy than can be accounted for by each individual single mutation.
The ability to carry out specific C-terminal modification or labeling of peptides and proteins has a broad range of applications. It is well established that this may be achieved by protease-catalyzed transacylation reactions and that carboxypeptidase Y (CPD-Y) is suitable for this due to its broad specificity and stability in the presence of denaturants. Furthermore, CPD-Y is characterized by a S'1 binding site that is open to solvent and, thus, capable of catalyzing a transpeptidation reaction with nucleophiles that extend beyond the perimeter of the active site. However, one major drawback with CPD-Y is that the yield of the reaction is highly dependent on the nature of the leaving group; e.g., with large apolar leaving groups the yield of the reaction does not exceed 15%. In the present publication it is demonstrated that mutants of CPD-Y, designed for low leaving group dependence, efficiently incorporate biocytin amide as well as a new fluorescent nucleophile, N'-Abz-Lysine amide (ablysin amide), into peptides and proteins.
The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.
The activity of serine carboxypeptidases is dependent on a catalytic triad, an oxyanion hole, and a binding site equivalent to those found in the serine endopeptidases. The action of carboxypeptidase Y on substrates containing amino acids, alcohols, and amines as leaving groups is described. It is demonstrated that the features common to serine endopeptidases and carboxypeptidases are sufficient for hydrolysis of ester bonds. However, rapid hydrolysis of amide bonds is dependent on interactions between the C-terminal carboxylate group of the substrate and the C-terminal recognition site of the enzyme. Furthermore, on the basis of the pH dependencies of wild-type and mutant enzyme, combined with the ability of the enzyme to utilize binding energy to promote catalysis, alternative models for the high activity of carboxypeptidase Y at low pH are discussed. They describe how the catalytically essential histidine is maintained in its active deprotonated state through perturbation of its pKa value in the enzyme-substrate complex.
The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD-WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and -170 degrees. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 A resolution for data collected at room temperature. The -170 degrees C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 A resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S1 through S4 sites, while density is apparent only in S1 and S2 for antipain. In the S1 site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P1 phenylalanyl side-chain of chymostatin. The P1 arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S1 site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approximation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseus proteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness.
The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1.
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The P1 substrate preference of serine carboxypeptidases, as expressed by the Lys/Leu ratio, differs by up to 10(5)-fold. Predictions of the major determinants of this preference are made by correlating primary and tertiary structures to substrate preferences. In carboxypeptidase Y from yeast it is predicted that Trp312 constitutes such a determinant, reducing the P1 Lys/Leu substrate preference of this enzyme. The predictions are tested by the construction and kinetic characterization of ten mutant enzymes of carboxypeptidase Y. All of these enzymes exhibit changes in their P1 substrate preference. Generally, small decreases in activity (kcat/Km) are observed with substrates containing uncharged P1 side chains. With substrates containing acidic P1 side chains, i.e., FA-Glu-Ala-OH, the activity generally increases slightly, 7-fold in the case of W312K. The most dramatic effects of the Trp312 substitutions are observed with substrates containing basic P1 side chains, i.e., kcat/Km for the hydrolysis of Fa-Lys-Ala-OH with W312E has increased 1150-fold, exclusively as a result of increased kcat values. Similar results have previously been obtained by mutational substitution at position 178 of carboxypeptidase Y. The construction and kinetic characterization of position 178 + 312 double mutants demonstrate that the kinetic effects of substitutions at these two positions are not additive. The P1 Lys/Leu substrate preference of one double mutant, L178D + W312D, has changed 380,000-fold as compared to the wild type enzyme, and the overall P1 substrate preference of this enzyme closely resembles that of carboxypeptidase WII from wheat.
The structure of monomeric serine carboxypeptidase from Saccharomyces cerevisiae (CPD-Y), deglycosylated by an efficient new procedure, has been determined by multiple isomorphous replacement and crystallographic refinement. The model contains 3333 non-hydrogen atoms, all 421 amino acids, 3 of 4 carbohydrate residues, 5 disulfide bridges, and 38 water molecules. The standard crystallographic R-factor is 0.162 for 10,909 reflections observed between 20.0- and 2.8-A resolution. The model has rms deviations from ideality of 0.016 A for bond lengths and 2.7 degrees for bond angles and from restrained thermal parameters of 7.9 A2. CPD-Y, which exhibits a preference for hydrophobic peptides, is distantly related to dimeric wheat serine carboxypeptidase II (CPD-WII), which has a preference for basic peptides. Comparison of the two structures suggests that substitution of hydrophobic residues in CPD-Y for negatively charged residues in CPD-WII in the binding site is largely responsible for this difference. Catalytic residues are in essentially identical configurations in the two molecules, including strained main-chain conformational angles for three active site residues (Ser 146, Gly 52, and Gly 53) and an unusual hydrogen bond between the carboxyl groups of Glu 145 and Glu 65. The binding of an inhibitor, benzylsuccinic acid, suggests that the C-terminal carboxylate binding site for peptide substrates is Asn 51, Gly 52, Glu 145, and His 397 and that the "oxyanion hole" consists of the amides of Gly 53 and Tyr 147. A surprising result of the study is that the domains consisting of residues 180-317, which form a largely alpha-helical insertion into the highly conserved cores surrounding the active site, are quite different structurally in the two molecules. It is suggested that these domains have evolved much more rapidly than other parts of the molecule and are involved in substrate recognition.
A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure. We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position. The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues. There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178. A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue. When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position. The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Serine carboxypeptidases have the ability to hydrolyze peptides as well as peptide amides. Previously, it has been demonstrated that Asn51 and Glu145 (in the protonated form) each donate a hydrogen bond to the alpha-carboxylate of peptide substrate. It is here demonstrated by characterization of carboxypeptidase Y derivatives, mutationally altered at positions 51 and 145, that the same groups are involved in the interaction with the C-terminal carboxyamide group of peptide amides. Asn51 donates a hydrogen bond to the C = O group of the substrate, and Glu145 (in the charged form) accepts one from the NH2 group of the substrate. Thus, the ionic state of Glu145 is different when peptides are hydrolyzed as compared with when peptide amides are hydrolyzed. This explains why Km for the hydrolysis of peptides increases with pH, whereas it remains constant for peptide amides. As a consequence, kcat/Km for the hydrolysis of peptide amides is higher than for the hydrolysis of peptides at pH > 8. At physiological pH, peptides and peptide amides are hydrolyzed with rates of the same order of magnitude; this is in accordance with reports describing that serine carboxypeptidases are involved in the degradation of biologically active peptide amides.
A solid-phase assay for the complete subsite mapping of the active site of endoproteases has been developed. A library of resin-bound protease substrates was synthesized both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer type of resin that allows proteases to diffuse into the interior and perform their catalytic activity. Anthranilic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet mixing of the beads and parallel washing procedures. After treatment with subtilisin Carlsberg, fluorescing beads were collected and subjected to peptide sequencing, affording the preferred sequences, their cleavage bond, and a semiquantitative estimation of the turnover. A statistical distribution of preferred amino acids was obtained for each subsite. The result was compared with data from kinetic studies in solution.
The three-dimensional structure of (serine) carboxypeptidase Y suggests that the side chains of Trp49, Asn51, Glu65, and Glu145 could be involved in the recognition of the C-terminal carboxylate group of peptide substrates. The mutations Trp49-->Phe; Asn51-->Ala, Asp, Glu, Gln, Ser, or Thr; Glu65-->Ala; and Glu145-->Ala, Asp, Asn, Gln, or Ser have been performed. Enzymes with Ala at these positions were also produced as double and triple mutations. These mutations have only little effect on the esterase activity of the enzyme, consistent with the absence of a hydrogen bond acceptor in the P1' position of such substrates. On the other hand, removal of the hydrogen-bonding capacity by incorporation of Ala at any of these four positions results in reduced peptidase activity, in particular when Asn51 and Glu145 are replaced. The results are consistent with Trp49 and Glu65 orienting Asn51 and Glu145 by hydrogen bonds, such that these can function as hydrogen bond donors (Glu145 only in its protonated carboxylic acid form) with the C-terminal alpha-carboxylate group of the peptide substrate as acceptor. However, it appears that strong interactions are formed only in the transition state since the combined removal of Asn51 and Glu145 reduces kcat about 100-fold and leaves KM practically unchanged. The results obtained with enzymes in which Asn51 or Glu145 has been replaced with other residues possessing the capacity to donate a hydrogen bond demonstrate that there is no flexibility with respect to the nature of the hydrogen bond donor at position 145, whereas enzymes with Gln, Ser, or Thr at position 51 exhibit much higher activity than N51A, although none of them reaches the wild-type level. With carboxypeptidase Y as well as other serine carboxypeptidases the binding of peptide substrates in the ground state (KM) is adversely affected by an increase in pH. It is shown that deprotonation of a single ionizable group with a pKa of 4.3 on the enzyme is responsible for this pH effect. The results show that the group involved is either Glu65 or Glu145, the latter being the more probable. The effect of this ionization on KM is explained by charge repulsion between the carboxylate group of the substrate and that of Glu145, hence preventing substrate from binding.
Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.