Remington SJ

References (12)

Title : Peptide aldehyde complexes with wheat serine carboxypeptidase II: implications for the catalytic mechanism and substrate specificity - Bullock_1996_J.Mol.Biol_255_714
Author(s) : Bullock TL , Breddam K , Remington SJ
Ref : Journal of Molecular Biology , 255 :714 , 1996
Abstract : 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.
ESTHER : Bullock_1996_J.Mol.Biol_255_714
PubMedSearch : Bullock_1996_J.Mol.Biol_255_714
PubMedID: 8636973
Gene_locus related to this paper: wheat-cbp02

Title : Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y from Yeast. The Significance of Thr60 and Met398 in Hydrolysis and Aminolysis Reactions - Sorensen_1995_J.Am.Chem.Soc_117_5944
Author(s) : Sorensen SB , Raaschou-Nielsen M , Mortensen UH , Remington SJ , Breddam K
Ref : Journal of the American Chemical Society , 117 :5944 , 1995
Abstract : In (serine)carboxypeptidase Y, the flexible side chain of Met 398 forms one side of the Si' binding pocket and the beta -and gamma-carbon atoms of Thr60 form the opposite side. Met398 has been substituted with the residues Gly,Ala,Val,lie,Leu,Phe,and Tyr while Thr60 has been substituted with the residues Ala,Val,Leu,Met,Phe,and Tyr by site-directed mutagenesis,and the resulting enzymes have been characterized with respect to their Pi' substrate preferences using thes ubstrate series FA-Phe-Xaa-OH (Xaa=Gly,Ala,Val,orLeu) and FA-Ala-Yaa-OH (Yaa=Leu,Gin,Glu,Lys,or Arg). The results show that Met398 is much more important for transition state stabilization than Thr60 although itappears that the selected non bulky amino acid residue(Thr) at position 60 is important for high Kcat values. The results further suggest that bulky amino acid side chains at position 398 are able to adjust the size of the Si' pocket such that favorable interactions with the substrate can be obtained with even small Pi' side chains,e.g., Gly. Accordingly,the hydrolysis of substrates with bulky/hydrophobic Pi' side chains is less dependent on the nature of the amino acid residue at position 398 than that of a substrate with a non bulky Pi' sid echain.The three-dimensional structure oft hemutant enzymeE65A+E145A has been determined, and it provides support for the high mobility of the Met398 side chain. In transpeptidation reactions the substitutions at position 398 also influence the interactions between the binding pocket and the amino acid leaving group as well as the added nucleophile competing with water in the deacylation reaction.Much higher aminolysis was obtained with some of the mutant enzymes, presumably due to a changed accessibility of water to the acyl-enzyme intermediate while the nucleophile/leaving group isbound at the Si' binding site.
ESTHER : Sorensen_1995_J.Am.Chem.Soc_117_5944
PubMedSearch : Sorensen_1995_J.Am.Chem.Soc_117_5944
PubMedID:
Gene_locus related to this paper: yeast-cbpy1

Title : Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y - Stennicke_1994_Protein.Eng_7_911
Author(s) : Stennicke HR , Mortensen UH , Christensen U , Remington SJ , Breddam K
Ref : Protein Engineering , 7 :911 , 1994
Abstract : Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.
ESTHER : Stennicke_1994_Protein.Eng_7_911
PubMedSearch : Stennicke_1994_Protein.Eng_7_911
PubMedID: 7971953
Gene_locus related to this paper: yeast-cbpy1

Title : Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate - Mortensen_1994_Biochemistry_33_508
Author(s) : Mortensen UH , Remington SJ , Breddam K
Ref : Biochemistry , 33 :508 , 1994
Abstract : 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.
ESTHER : Mortensen_1994_Biochemistry_33_508
PubMedSearch : Mortensen_1994_Biochemistry_33_508
PubMedID: 7904479
Gene_locus related to this paper: yeast-cbpy1

Title : Structure of the complex of L-benzylsuccinate with wheat serine carboxypeptidase II at 2.0-A resolution - Bullock_1994_Biochemistry_33_11127
Author(s) : Bullock TL , Branchaud B , Remington SJ
Ref : Biochemistry , 33 :11127 , 1994
Abstract : The structure of the complex of L-benzylsuccinate (Ki = 0.2 mM) bound to wheat serine carboxypeptidase II has been analyzed at 2.0-A resolution for native and inhibited crystals at -170 degrees C. The model has been refined and has a standard crystallographic R-factor of 0.176 for 57,734 reflections observed between 20.0- and 2.0-A resolution. The root mean square deviation from ideal bonds is 0.017 A and from ideal angles is 2.6 degrees. The model consists of 400 amino acids, 4 N-linked saccharide residues, and 430 water molecules. L-Benzylsuccinate occupies a narrow slot in the active site defined by Tyr 60, Tyr 239, and the polypeptide backbone. One carboxylate forms hydrogen bonds to Glu 145, Asn 51, the amide of Gly 52, and the catalytic His 397, suggestive of how the peptide C-terminal carboxylate is recognized by the enzyme. The phenyl ring stacks between Tyr 239 and Tyr 60, while the other carboxylate occupies the "oxyanion hole". One of the oxygens accepts hydrogen bonds from the amides of Tyr 147 and Gly 53, while the other forms a very close contact (2.3 A) with the O gamma of Ser 146, forcing the side chain into a conformation alternative to that found in the resting state of the enzyme. The inhibitor occupies the active site in a way that suggests that it can be regarded as a transition-state analogue of serine carboxypeptidases. The model suggests a novel enzymatic mechanism, involving substrate-assisted catalysis, that might account for the low pH optimum (4.0-5.5) of peptidase activity unique to this family of serine proteinases.
ESTHER : Bullock_1994_Biochemistry_33_11127
PubMedSearch : Bullock_1994_Biochemistry_33_11127
PubMedID: 7727364
Gene_locus related to this paper: wheat-cbp02

Title : 2.8-A structure of yeast serine carboxypeptidase - Endrizzi_1994_Biochemistry_33_11106
Author(s) : Endrizzi JA , Breddam K , Remington SJ
Ref : Biochemistry , 33 :11106 , 1994
Abstract : 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.
ESTHER : Endrizzi_1994_Biochemistry_33_11106
PubMedSearch : Endrizzi_1994_Biochemistry_33_11106
PubMedID: 7727362
Gene_locus related to this paper: yeast-cbpy1

Title : The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178 - Olesen_1994_Biochemistry_33_11121
Author(s) : Olesen K , Mortensen UH , Aasmul-Olsen S , Kielland-Brandt MC , Remington SJ , Breddam K
Ref : Biochemistry , 33 :11121 , 1994
Abstract : 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)
ESTHER : Olesen_1994_Biochemistry_33_11121
PubMedSearch : Olesen_1994_Biochemistry_33_11121
PubMedID: 7727363

Title : The primary structure of carboxypeptidase S1 from Penicillium janthinellum - Svendsen_1993_FEBS.Lett_333_39
Author(s) : Svendsen I , Hofmann T , Endrizzi J , Remington SJ , Breddam K
Ref : FEBS Letters , 333 :39 , 1993
Abstract : The complete amino acid sequence of carboxypeptidase S1 from Penicillium janthinellium has been determined by N-terminal sequencing of the reduced and vinylpyridinated protein and of peptides obtained by cleaved with cyanogen bromide, iodosobenzoic acid, hydroxylamine, endoproteinase LysC, endoproteinase AspN and Glu-specific proteinase from B. licheniformis. The enzyme consists of a single peptide chain of 433 amino acid residues and contains 9 half-cystine residues and one glycosylated asparagine residue. A comparison to other carboxypeptidases shows that the enzyme is homologous to carboxypeptidase-Y and carboxypeptidase-MIII from malt. Specificity and binding of substrates are discussed from a three-dimensional model based on the known structure of carboxypeptidase-Y from Saccharomyces cereviciae and carboxypeptidase II from wheat.
ESTHER : Svendsen_1993_FEBS.Lett_333_39
PubMedSearch : Svendsen_1993_FEBS.Lett_333_39
PubMedID: 8224168
Gene_locus related to this paper: penja-cps1

Title : Refined atomic model of wheat serine carboxypeptidase II at 2.2-A resolution - Liao_1992_Biochemistry_31_9796
Author(s) : Liao DI , Breddam K , Sweet RM , Bullock T , Remington SJ
Ref : Biochemistry , 31 :9796 , 1992
Abstract : The crystal structure of the homodimeric serine carboxypeptidase II from wheat (CPDW-II, M(r) 120K) has been determined and fully refined at 2.2-A resolution to a standard crystallographic R factor of 16.9% using synchrotron data collected at the Brookhaven National Laboratory. The model has an rms deviation from ideal bond lengths of 0.018 A and from bond angles of 2.8 degrees. The model supports the general conclusions of an earlier study at 3.5-A resolution and will form the basis for investigation into substrate binding and mechanistic studies. The enzyme has an alpha + beta fold, consisting of a central 11-stranded beta-sheet with a total of 15 helices on either side. The enzyme, like other serine proteinases, contains a "catalytic triad" Ser146-His397-Asp338 and a presumed "oxyanion hole" consisting of the backbone amides of Tyr147 and Gly53. The carboxylate of Asp338 and imidazole of His397 are not coplanar in contrast to the other serine proteinases. A comparison of the active site features of the three families of serine proteinases suggests that the "catalytic triad" should actually be regarded as two diads, a His-Asp diad and a His-Ser diad, and that the relative orientation of one diad with respect to the other is not particularly important. Four active site residues (52, 53, 65, and 146) have unfavorable backbone conformations but have well-defined electron density, suggesting that there is some strain in the active site region. The binding of the free amino acid arginine has been analyzed by difference Fourier methods, locating the binding site for the C-terminal carboxylate of the leaving group. The carboxylate makes hydrogen bonds to Glu145, Asn51, and the amide of Gly52. The carboxylate of Glu145 also makes a hydrogen bond with that of Glu65, suggesting that one or both may be protonated. Thus, the loss of peptidase activity at pH > 7 may in part be due to deprotonation of Glu145. The active site does not reveal exposed peptide amides and carbonyl oxygen atoms that could interact with substrate in an extended beta-sheet fashion. The fold of the polypeptide backbone is completely different than that of trypsin or subtilisin, suggesting that this is a third example of convergent molecular evolution to a common enzymatic activity. Furthermore, it is suggested that the active site sequence motif "G-X-S-X-G/A", often considered the hallmark of serine peptidase or esterase activity, is fortuitous and not the result of divergent evolution.
ESTHER : Liao_1992_Biochemistry_31_9796
PubMedSearch : Liao_1992_Biochemistry_31_9796
PubMedID: 1390755
Gene_locus related to this paper: wheat-cbp02

Title : The alpha\/beta hydrolase fold - Ollis_1992_Prot.Engin_5_197
Author(s) : Ollis DL , Cheah E , Cygler M , Dijkstra B , Frolow F , Franken SM , Harel M , Remington SJ , Silman I , Schrag JD , Sussman JL , Verschueren KHG , Goldman A
Ref : Protein Engineering , 5 :197 , 1992
Abstract : We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
ESTHER : Ollis_1992_Prot.Engin_5_197
PubMedSearch : Ollis_1992_Prot.Engin_5_197
PubMedID: 1409539

Title : Crystallization of serine carboxypeptidases - Wilson_1990_J.Mol.Biol_211_301
Author(s) : Wilson KP , Liao DI , Bullock T , Remington SJ , Breddam K
Ref : Journal of Molecular Biology , 211 :301 , 1990
Abstract : Crystallization of three different serine carboxypeptidases has been achieved by the method of hanging-drop vapor diffusion. Serine carboxypeptidases II from wheat bran and malted barley crystallize isomorphously from polyethylene glycol solutions at room temperature (pH 4 to 7) in space group P4(1)2(1)2 or enantiomorph with cell dimensions of a = b = 98.2 A and c = 209.5 A. The crystals diffract to about 2.3 A resolution using rotating-anode X-ray generators. Assuming a dimer of Mr 120,000 in the asymmetric unit, Vm = 2.1 A3/dalton. These crystals appear suitable for structural studies. A genetically engineered serine carboxypeptidase from yeast, which lacks three of four glycosylation sites present in the wild-type, has also been crystallized by vapor diffusion against methylpentanediol at 4 degrees C, pH 6.4 to 8.0.
ESTHER : Wilson_1990_J.Mol.Biol_211_301
PubMedSearch : Wilson_1990_J.Mol.Biol_211_301
PubMedID: 2308160
Gene_locus related to this paper: wheat-cbp02

Title : Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase - Liao_1990_J.Biol.Chem_265_6528
Author(s) : Liao DI , Remington SJ
Ref : Journal of Biological Chemistry , 265 :6528 , 1990
Abstract : The structure of serine carboxypeptidase II from wheat bran has been determined to 3.5-A resolution by multiple isomorphous replacement, solvent flattening, and crystallographic refinement. The amino acid sequence has been fit to the electron density map and the model refined to a conventional crystallographic R factor of 20.9%. The molecule is an alpha + beta protein and contains a "catalytic triad" (Asp338, His397, and Ser146) similar in arrangement to those in chymotrypsin and subtilisin. The -fold of the polypeptide backbone is, however, completely different from those enzymes. This suggests that this is a third example of convergent evolution to a common enzymatic mechanism. The -fold is, on the other hand, surprisingly similar to that of the zinc proteinase carboxypeptidase A.
ESTHER : Liao_1990_J.Biol.Chem_265_6528
PubMedSearch : Liao_1990_J.Biol.Chem_265_6528
PubMedID: 2324088
Gene_locus related to this paper: wheat-cbp02