Identification of the catalytic histidine residue participating in the charge-relay system of carboxypeptidase Y.

The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N tau-carboxy-methyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO2-Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participate in the charge-relay system of CPY.     

羧肽酶Y标记蛋白质羧基端方法的优化及应用

蛋白质水解是一种重要的翻译后修饰,它在许多生化过程 (如细胞凋亡和肿瘤细胞转移等) 中起着极其重要的作用。鉴定蛋白质水解位点可以进一步加深我们对这些生化过程的认识。尽管蛋白质氨基端标记方法和蛋白质组学在复杂生物体系中鉴定获得了许多蛋白质的水解位点,但这种方法存在固有的缺陷。羧基端标记方法是另一种可行的鉴定蛋白质水解位点的方法。本文优化了蛋白质羧基端生物酶标记方法,提高了亲和标记效率,从而可以更好地利用正向分离方法对蛋白质羧基端多肽进行分离并用质谱鉴定。我们用优化后的羧基端标记方法来标记大肠杆菌Escherichia coli复杂蛋白样品后鉴定到了120多个蛋白质羧基端多肽和内切多肽。在其所鉴定的蛋白质水解位点中,我们发现了许多已知和未知的位点,这些新的水解位点有可能在正常生化过程的调控发挥着重要的作用。该研究提供了一个可以与蛋白质氨基端组学互为补充、可在复杂体系中鉴定蛋白质水解的方法。     

Structural principles of the wide substrate specificity of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis> carboxypeptidase T. reconstruction of the carboxypeptidase B primary specificity pocket

Site-directed mutagenesis in the active site of Thermoactinomyces vulgaris carboxypeptidase T (CpT), which is capable of hydrolyzing both hydrophobic and positively charged substrates, resulted in five mutants: CpT1 (A243G), CpT2 (D253G/T255D), CpT3 (A243G/D253G/T255D), CpT4 (G207S/A243G/D253G/T255D), and CpT5 (G207S/A243G/T250A/D253G/T255D). These mutants step-by-step reconstruct the primary specificity pocket of carboxypeptidase B (CpB), which is capable of cleaving only positively charged C-terminal residues. All of the mutants retained the substrate specificity of the wild-type CpT. Based on comparison of three-dimensional structures of CpB and the CpT5 model, it was suggested that the lower affinity of CpT5 for positively charged substrates than the affinity of CpB could be caused by differences in nature and spatial location of Leu247 and Ile247 and of His68 and Asp65 residues in CpT and CpB, respectively, and also in location of the water molecule bound with Ala250. An additional hydrophobic region was detected in the CpT active site formed by Tyr248, Leu247, Leu203, Ala243, CH3-group of Thr250, and CO-groups of Tyr248 and Ala243, which could be responsible for binding hydrophobic substrates. Thus, notwithstanding the considerable structural similarity of CpT and pancreatic carboxypeptidases, the mechanisms underlying their substrate specificities are different.     

Inhibition of carboxypeptidase A by excess zinc: analysis of the structural determinants by X-ray crystallography

Pancreatic metallocarboxypeptidases are inhibited by a millimolar excess of zinc together with other exo- and endometalloproteases. We have analyzed the structure of bovine carboxypeptidase A inhibited by an excess of zinc ions using X-ray crystallography at 1.7 Å overall resolution. Under these conditions, a second zinc is observed to bind to the enzyme active site, establishing a distorted tetrahedrally coordinated complex which involves Glu-270 (the general base for catalysis), a water molecule, a chloride ion, and a hydroxide ion. This hydroxide ion forms a 114° angular bridge between the inhibitory and the catalytic zinc ions, which are at a distance of 3.3 Å from one another. The inhibitory zinc holds the hydroxide at nearly the same location as a previously observed active site water molecule (W571) and probably perturbs the substrate positioning and stereochemical rearrangements required for substrate cleavage during catalysis.     

Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts.

Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d-phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 A centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 A sphere of water molecules. MD calculations were carried out for 45-60 ps. There is no large deviation from the available X-ray structures of native and the dPhe adduct for the MD structures. Average MD structures were calculated starting from the X-ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed.     

Crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 A. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild-type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis.     

A rational approach for the development of reduced-size analogues of neuropeptide Y with high affinity to the Y1 receptor

Four sets of centrally truncated analogues of neuropeptide Y have been synthesized. In each series the N-terminal part was constant, while the C-terminal segment was systematically varied in length. The C- and N-terminal parts were linked by 6-aminohexanoic acid. The affinity to the Y₁ receptor was investigated on human neuroblastoma cells SK-N-MC. Significant differences were found between the series of peptides as well as within each set. Remarkably, the affinity did not solely depend on the length of the segment, and with increasing numbers of residues the IC₅₀ values were not always decreased. With a given N-terminal segment, only one optimal length of the C-terminal segment was found, which suggests that it is not the amino acids themselves but their 3D arrangement and orientation that is important for high receptor affinity.     

Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

The prevalence of aromatic residues in the ligand binding site of the GABA_A receptor, as with other cys‐loop ligand‐gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β₂Tyr97, β₂Tyr157, and β₂Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK‐293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA_A receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β₂Tyr157 and β₂Tyr205 are more detrimental than β₂Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.     

The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.     

Conserved water molecules in the specificity pocket of serine proteases and the molecular mechanism of Na+ binding

Conservation of clusters of buried water molecules is a structural motif present throughout the serine protease family. Frequently, these clusters are shaped as water channels forming extensive hydrogen-bonding networks linked to the protein backbone. The most conspicuous example is the water channel present in the specificity pocket of trypsin and thrombin. In thrombin, other vitamin K-dependent proteases, and some complement factors, Na⁺ binds in this water channel and enhances allosterically the catalytic activity of the enzyme, whereas digestive and fibrinolytic proteases are devoid of such regulation. A comparative analysis of proteases with and without Na⁺ binding capability reveals the role of the water channel in maintaining the structural organization of the specificity pocket and in Na⁺ coordination. This enables the formulation of a molecular mechanism for Na⁺ binding in thrombin and leads to the identification of the structural changes necessary to engineer a functional Na⁺ site and enhanced catalytic activity in trypsin and other proteases. Proteins 30:34–42, 1998. © 1998 Wiley-Liss, Inc.     

The association between a negatively charged ligand and the electronegative binding pocket of its receptor.

Many examples exist of charged amino acids that play a role in attracting or holding a charged ligand toward or inside an oppositely charged binding pocket of the protein. For example, the enzymes superoxide dismutase, triose-phosphate isomerase, and acetylcholinesterase can steer ligands toward their oppositely charged binding pockets or gorges. Interestingly, in our Brownian dynamics simulations of a phosphate-binding protein, we discovered that negatively charged phosphate (HPO(2-)(4)) could make its way into the negatively charged binding pocket. In fact, the phosphate-binding protein exhibits counterintuitive kinetics of association. That is, one would expect that the rate of association would increase on increases to the ionic strength since the interaction between the ligand, with a charge of -2, and the electronegative binding pocket would be repulsive and greater screening should reduce this repulsion and increase the rate of association. However, the opposite is seen-i.e., the rate of association decreases on increases in the ionic strength. We used Brownian dynamics techniques to compute the diffusion limited association rate constants between the negatively charged phosphate ligand and several open forms of PBP (wild-type and several mutants based on an x-ray structure of open-form PBP, mutant T141D). With the appropriate choices of reaction criteria and molecular parameters, the ligand was able to diffuse into the binding pocket. A number of residues influence binding of the ligand within the pocket via hydrogen bonds or salt bridges. Arg135 partially neutralizes the charges on the HPO(2-)(4) ligand in the binding pocket, allowing it to enter. It is also found that the positive electrostatic patches above and below the binding entrance of PBP contribute the major attractive forces that direct the ligand toward the surface of the protein near the binding site.     

Phylogenetic and mutational analyses reveal key residues for UDP-glucuronic acid binding and activity of beta1,3-glucuronosyltransferase I (GlcAT-I)

The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.     

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.     

The catabolite gene activation system of E. coli may be directly involved in regulation of bacteriophage lambda development

The primary structure of bacteriophage lambda DNA has been searched for the presence of consensus CAP binding sites. Four putative CAP binding sites have been found on the lambda genome, indicating that the catabolite gene activation system of E. coli may be directly involved in the regulation of lambda development. Molecular mechanisms of putative cAMP-CAP-mediated stimulation of lysogenic and lytic responses are discussed.     

Evolutionary trace analysis at the ligand binding site of laccase

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.     

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Proposed structure of the F' allotype of human CR1. Loss of a C3b binding site may be associated with altered function.

Human CR1 is composed of tandem long homologous repeating (LHR) segments that encode separate binding sites for C3b or C4b. Homologous recombination with unequal crossover has been proposed as the genetic mechanism that gave rise to the CR1 alleles that differed in their total numbers of LHR. The F allotype has four LHR, named LHR-A, -B, -C, -D, 5' to 3'. The site in LHR-A preferentially binds C4b and those in LHR-B and -C prefer C3b. A previous study revealed the presence of a fifth LHR with sequences similar to LHR-B and a third C3b binding site in the S allotype of higher m.w. In the present study, an 18-kb EcoRV fragment that was associated with the expression of the lower m.w. F' allotype hybridized with a unique pattern of cDNA and intron probes specific for LHR-C. Deletion of LHR-B and one C3b binding site was proposed as the mechanism for the appearance of this F'-specific fragment. Functional differences among the CR1 variants were sought by comparative analyses of soluble rCR1 having one, two or three C3b binding sites. Although these three variants did not exhibit any significant differences in their capacities to act as cofactors for the cleavage of monomeric C3b, their relative affinities for dimeric ligand varied more than 100-fold. Furthermore, the variant with only one C3b binding site was at least 10-fold less effective in the inhibition of the alternative pathway C3 and C5 convertases. These observations suggested that the F' allotype may be impaired in its capacity to bind opsonized immune complexes, to inhibit the formation of the alternative pathway C3 and C5 convertases, and perhaps to mediate other CR1-dependent cellular responses.     

Adsorption and infectivity of human immunodeficiency virus type 1 are modified by the fluidity of the plasma membrane for multiple-site binding

Based on the assumption that fluidity of the plasma membrane and viral envelope is necessary for recruiting additional receptors and ligands to the initial attachment site for "multiple-site binding," we determined the effect of increased temperature on viral infectivity. Infection of human immunodeficiency virus type 1 (HIV-1) and a pseudotyped luciferase-expressing chimeric virus using MAGI and GHOST/CXCR4 cells showed that in 1 hr of viral adsorption the extent of virus infection and the amount of tightly adsorbed viruses depended on temperature; and that membrane fluidity increased according to increased temperature. Augmented infection was observed as post-attachment enhancement (PAE) when cells were washed and incubated at 40 C for 1 hr after viral adsorption. PAE was completely inhibited by 1 micro M of anti-CXCR4 peptide T140, and addition of T140 at 20 min resulted in a gradual loss of inhibition of PAE, indicating the need for a 30 to 40 min timelag to ensure tight multiple-site binding. These data suggest that the accumulation of gp120 and receptor complex (multiple-site binding) was needed to complete the infection. Treatments of cells with 0.05% Tween 20 or 2 micro g/ml of anti-HLA-II antibody resulted in increases or decreases, respectively, of attached viruses and the infectivity. As well, Tween 20 and anti-HLAII antibody enhanced and suppressed the fluidity of the plasma membrane, respectively. Amounts of adsorbed viruses and degrees of viral infectivity correlated with the intensity of fluidity of the plasma membrane, probably due to the formation of multiple-site binding.