Biomedical subjects

John W Kozarich

Publications and source records attributed to John W Kozarich.

10 recordsLinked to original sources

PubMed2007

Functional interrogation of the kinome using nucleotide acyl phosphates.

The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.

Adenine Nucleotides↗

PubMed2005

Synthesis and structure-activity relationship of N-alkyl Gly-boro-Pro inhibitors of DPP4, FAP, and DPP7.

The structure-activity relationship of various N-alkyl Gly-boro-Pro derivatives against three dipeptidyl peptidases (DPPs) was studied. In a series of N-cycloalkyl analogs, DPP4 and fibroblast activation protein-alpha (FAP) optimally preferred N-cycloheptyl whereas DPP7 tolerated even larger cycloalkyl rings. Gly alpha-carbon derivatization of N-cyclohexyl or N-(2-adamantyl) Gly-boro-Pro resulted in a significant decrease in potency against all the three DPPs.

Adenosine Deaminase Inhibitors↗

PubMed2005

Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors.

Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM.

Boronic Acids↗

PubMed2004

Crystal structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida, a fumarase class II type cycloisomerase: enzyme evolution in parallel pathways.

3-Carboxy-cis,cis-muconate lactonizing enzymes (CMLEs), the key enzymes in the protocatechuate branch of the beta-ketoadipate pathway in microorganisms, catalyze the conversion of 3-carboxy-cis,cis-muconate to muconolactones. We have determined the crystal structure of the prokaryotic Pseudomonas putida CMLE (PpCMLE) at 2.6 A resolution. PpCMLE is a homotetramer and belongs to the fumarase class II superfamily. The active site of PpCMLE is formed largely by three regions, which are moderately conserved in the fumarase class II superfamily, from three respective monomers. It has been proposed that residue His141, which is highly conserved in all fumarase class II enzymes and forms a charge relay with residue Glu275 (both His141 and Glu275 are in adenylosuccinate lyase numbering), acts as the general base in most fumarase class II superfamily members. However, this charge relay pair is broken in PpCMLE. The residues corresponding to His141 and Glu275 are Trp153 and Ala289, respectively, in PpCMLE. The structures of prokaryotic MLEs and that of CMLE from the eukaryotic Neurospora crassa are completely different from that of PpCMLE, indicating MLEs and CMLEs, as well as the prokaryotic and eukaryotic CMLEs, evolved from distinct ancestors, although they catalyze similar reactions. The structural differences may be related to recognition by substrates and to differences in the mechanistic pathways by which these enzymes catalyze their respective reactions.

Amino Acid Sequence↗

PubMed2004

Mapping enzyme active sites in complex proteomes.

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.

Aldehyde Dehydrogenase↗

PubMed2003

Activity-based proteomics: enzyme chemistry redux.

The principles of enzyme chemistry, mechanism of action and inhibitor design are being applied to proteomics by the development of activity-based probes. This approach suggests a potentially broad method for interrogating enzyme family members, both known and unknown, in cells and proteomic fractions without the need for individual assay development and isolation. The serine hydrolases and cysteine proteases have provided the proofs of concept for activity-based proteomics, and other studies are rapidly following. The result will be a proteomics technology of great value to drug discovery and development.

Enzyme Inhibitors↗

PubMed2003

Prolyl peptidases: a serine protease subfamily with high potential for drug discovery.

Much attention has recently been given to a class of proteases that cleave proteins and peptides after proline residues. This class includes dipeptidyl peptidase IV (DPP IV; also termed CD26), fibroblast activation protein alpha (FAP; seprase), DPP7 (DPP II; quiescent cell proline dipeptidase), DPP8, DPP9, and prolyl carboxypeptidase (PCP; angiotensinase C). More distant members include prolyl oligopeptidase (POP; post proline cleaving enzyme) and acylaminoacylpeptidase (AAP; acylpeptide hydrolase). The DPPs and related proteins contain both membrane-bound and soluble members and span a broad range of expression patterns, tissue distributions and compartmentalization. These proteins have important roles in regulation of signaling by peptide hormones, and are emerging targets for diabetes, oncology and other indications.

Animals↗

PubMed2002

Structure of Escherichia coli pyruvate formate-lyase with pyruvate.

The structure of inactive pyruvate formate-lyase in complex with a natural substrate, pyruvate, was solved at 2.7 A resolution. Both active sites of the homodimeric enzyme are occupied by pyruvate; additional binding sites were not found. Pyruvate was found in a cleft close to the active-site cysteines 418 and 419, with the carboxyl group in contact with arginines 176 and 435 and the methyl group within van der Waals distance of Phe327. It is believed that the binding site of pyruvate is not the position of pyruvate as the reaction initiates, as conformational changes occur during activation of the enzyme.

Acetyltransferases↗

PubMed2002

Solution structure of the hydroperoxide of Co(III) phleomycin complexed with d(CCAGGCCTGG)2: evidence for binding by partial intercalation.

The bleomycins (BLMs) are natural products that in the presence of iron and oxygen bind to and cause single-strand and double-strand cleavage of DNA. The mode(s) of binding of the FeBLMs that leads to sequence-specific cleavage at pyrimidines 3' to guanines and chemical-specific cleavage at the C-4' H of the deoxyribose of the pyrimidine has remained controversial. 2D NMR studies using the hydroperoxide of CoBLM (HOO-CoBLM) have demonstrated that its bithiazole tail binds by partial intercalation to duplex DNA. Studies with ZnBLM demonstrate that the bithiazole tail binds in the minor groove. Phleomycins (PLMs) are BLM analogs in which the penultimate thiazolium ring of the bithiazole tail is reduced. The disruption of planarity of this ring and the similarities between FePLM- and FeBLM-mediated DNA cleavage have led Hecht and co-workers to conclude that a partial intercalative mode of binding is not feasible. The interaction of HOO-CoPLM with d(CCAGGCCTGG)2 has therefore been investigated. Binding studies indicate a single site with a K(d) of 16 micro M, 100-fold greater than HOO-CoBLM for the same site. 2D NMR methods and molecular modeling using NMR-derived restraints have led to a structural model of HOO-CoPLM complexed to d(CCAGGCCTGG)2. The model reveals a partial intercalative mode of binding and the basis for sequence specificity of binding and chemical specificity of cleavage. The importance of the bithiazoles and the partial intercalative mode of binding in the double-strand cleavage of DNA is discussed.

Base Sequence↗

PubMed2002

The structure of Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme, a beta propeller cycloisomerase.

Muconate lactonizing enzymes (MLEs) convert cis,cis-muconates to muconolactones in microbes as part of the beta-ketoadipate pathway; some also dehalogenate muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme (NcCMLE) at 2.5 A resolution, with a seven-bladed beta propeller fold. It is related neither to bacterial MLEs nor to other beta propeller enzymes, but is structurally similar to the G protein beta subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and NcCMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. NcCMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.

Amino Acid Sequence↗