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Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog.

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 Å structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the α/β-hydrolase domain and an eight-bladed β-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp181, Lys120, and Tyr46. PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Dipeptidyl peptidases 8 and 9 : specificity and molecular characterization compared with dipeptidyl peptidase IV

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.

Crystallization and preliminary X-ray diffraction studies of an alkaline protease from Bacillus lentus

Abstract Various crystal forms of the subtilisin-type protease Savinase (EC 3.4.21.14) from the alkalophilic bacterium Bacillus lentus have been obtained. The first were orthorhombic needles, space group P212121, with unit cell dimensions a = 75.3 A , b = 53.4 A , c = 61.5 A . The crystals diffract to at least 1.8 A resolution, and the data to 2.0 A have been recorded on film using synchrotron radiation. The second crystal form grows as similar orthorhombic needles, also in P212121, with cell dimensions a = 75.5 A , b = 47.4 A , c = 62.5 A , mainly differing from the first in the shorter b-axis. Data have been recorded to 2.8 A. The third form is monoclinic, space group P21 with dimensions a = 40.7 A , b = 64.4 A , c = 43.0 A , β = 119 ° . Data to a spacing of 2.4 A have been recorded for this form.

Enzyme kinetic characterization of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta, PTPepsilon, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPbeta with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPalpha-D1 and PTPalpha-D1D2 suggest that D2 affects the catalysis of pNPP. PTPepsilon and the closely homologous PTPalpha behave differently. The K(m) data were lower for PTPepsilon than those for PTPalpha, while the inverse was observed for the catalytic efficiencies.

X-Ray structure of the antibiotic bacitracin A☆

Bacitracins are a group of widely used peptide antibiotics. There has been interest in determining the three‐dimensional structure of the bacitracins. However, solution studies indicate significant flexibility in their structure and to date native bacitracins have resisted attempts at crystallisation despite considerable efforts over a number of years by several groups. Here we report the first three‐dimensional X‐ray structure of a bacitracin, complexed to a subtilisin proteinase. X‐Ray diffraction data were collected using synchrotron radiation in combination with the Image Plate Scanner system. The complex structure including two enzymes, two bacitracins, 220 water molecules and two Ca2+ ions was refined by restrained least‐squares to a crystallographic R factor (=Σ{{F o‐F c}}/Σ{F o}}) of 16.3% at 2.0 Å.

Introduction of a free cysteinyl residue at position 68 in the subtilisin Savinase, based on homology with proteinase K

Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this ‐SH group a cysteinyl residue has been introduced by site‐directed mutagenesis into the cysteine‐free subtilisin‐like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the ‐SH group is not involved in catalysis.

An Investigation of the Savinase Water Channel: Implications of Cavity Mutations

The interior packing of proteins is believed to play a crucial role in stabilizing a protein’s conformation. Recently, the influence of changes in the interior packing on thermostability and function of proteins have been examined by means of protein engineering. Hydrophobic residues were substituted by more bulky residues in T4 lysozyme to fill a large cavity1,2 in order to improve the interior packing. Crystal structure analysis of the mutant proteins showed that the introduced aminoacids are accommodated with little perturbation of the three dimensional structures. The mutant proteins had normal activities and their thermal stabilities were marginally lower than those of the wildtype protein. Although the incorporation of more bulky hydrophobic sidechains in the core is expected to result in an increase in hydrophobic (entropic) stabilization a reduction in stability was observed. This result was ascribed to introduction of strain in the form of non-optimal dihedral angles, bond angle distortion and unfavorable van der Waals contacts (a positive free energy contribution).

PH Dependence of the Catalytic Activity of a Subtilisin-Like Proteinase

Three-dimensional crystal structures of serine proteinases1-4 have been mainly determined between pH 4 and 7 for both trypsin-like and subtilisin-like enzymes. No structures are available for the subtilisins, even the alkalophilic ones, around the most active pH range 9–11. This is the case for the highly alkalophilic enzyme, savinase, which was studied at pH 65. We have analysed two mutants of savinase which have isomorphous crystal forms under the following pH conditions: (1) mutant 19 at pH 10.5, where the protein is active and (2) mutant 17 at pH 6 where activity drops to less than 20%5. The mutations of the wild type structure are H120D, G195E and K235E for mutant 17, and H120D, R170Y, G195E and K235E for 19. Thus the two mutants differ from one another only by a single point mutation, R170Y. All these mutations are far from the active site in positions which should not affect the part of the structure important for the observations below. From these studies a direct comparison of the active and inactive structures has been made. The substrate binding site is a cleft or channel on the surface of the enzyme. The sides of the channel are made up of two s-strands of the enzyme, residues 95–103 and 123–134. In the native protein these are not part of a s-sheet. When substrate or peptide inhibitor binds, it occupies the channel so as to form a three stranded s-sheet with these two strands.