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George L Kenyon

Publications and source records attributed to George L Kenyon.

18 recordsLinked to original sources
PubMed2006

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase.

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2006

Exploring the role of the active site cysteine in human muscle creatine kinase.

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.

Adenosine Diphosphate↗
PubMed2006

Heterogeneity of Escherichia coli-expressed human muscle creatine kinase.

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.

Cloning, Molecular↗
PubMed2005

Exploring the active site of benzaldehyde lyase by modeling and mutagenesis.

Benzaldehyde lyase (BAL) is a thiamin diphosphate-dependent enzyme, which catalyzes the breakdown of (R)-benzoin to benzaldehyde. In essence, this is the reverse of the carboligation reaction catalyzed by benzoylformate decarboxylase (BFD). Here, we describe the first steps towards understanding the factors influencing BFD to form a CC bond under conditions wherein BAL will cleave the same bond. What are the similarities and differences between these two enzymes that result in the different catalytic activities? The X-ray structures of BFD and pyruvate decarboxylase (PDC) were used as templates for modeling benzaldehyde lyase. The model shows that a glutamine residue, Gln113, replaces the active site histidines of BFD and PDC. Replacement of the Gln113 by alanine or histidine reduced the value of k(cat) for lyase activity by more than 200-fold. The residues in BFD interacting with the phenyl ring of benzoylformate have similarly positioned counterparts in BAL but Ser26, the residue known to interact with the carboxylate group of benzoylformate, has been replaced by an alanine (Ala28). The BAL A28S variant exhibited 7% of WT activity in the BAL assay but, in the most intriguing result, this variant was able to catalyze the decarboxylation of benzoylformate. Conversely, the BFD S26A variant was unable to cleave benzoin.

Aldehyde-Lyases↗
PubMed2005

Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida.

Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.

Amino Acid Sequence↗
PubMed2004

Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase.

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by ATP. From a structural perspective, the enzyme utilizes two flexible loop regions to sequester and position the substrates for catalysis. There has been debate over the specific roles of the flexible loops in substrate specificity and catalysis in CK and other related phosphagen kinases. In CK, two hydrophobic loop residues, I69 and V325, make contacts with the N-methyl group of creatine. In this study, we report the alteration of the substrate specificity of CK through the mutagenesis of V325. The V325 to glutamate mutation results in a more than 100-fold preference for glycocyamine, while mutation of V325 to alanine results in a slight preference of the enzyme for cyclocreatine (1-carboxymethyl-2-iminoimidazolidine). This study enhances our understanding of how the active sites of phosphagen kinases have evolved to recognize their respective substrates and catalyze their reactions.

Amino Acid Sequence↗
PubMed2004

Novel mechanism of inhibition of HIV-1 reverse transcriptase by a new non-nucleoside analog, KM-1.

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.

Adenosine Triphosphate↗
PubMed2004

Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family.

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.

Amidohydrolases↗
PubMed2003

Mechanism-based inactivation of thioredoxin reductase from Plasmodium falciparum by Mannich bases. Implication for cytotoxicity.

Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is an essential protein which has been validated as a drug target. The high-throughput screening of 350000 compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agents leading to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis, different Mannich bases possessing one or two electrophilic centers were synthesized and first studied in detail using glutathione as a model thiol. Michael addition of glutathione to the double bond of an unsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promoted by base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. The intermediate formed in this reaction is an alpha,beta-unsaturated ketone that can react rapidly with a second thiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can be observed spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turn over its natural substrate Trx, it can reduce low M(r) electron acceptors such as methyl methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases, a detailed mechanism for the inactivation of TrxR is proposed.

Alkylating Agents↗
PubMed2003

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase.

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.

Alanine↗
PubMed2003

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.

Aldehyde Oxidoreductases↗
PubMed2002

The 2.1 A structure of Torpedo californica creatine kinase complexed with the ADP-Mg(2+)-NO(3)(-)-creatine transition-state analogue complex.

Creatine kinase (CK) catalyzes the reversible conversion of creatine and ATP to phosphocreatine and ADP, thereby helping maintain energy homeostasis in the cell. Here we report the first X-ray structure of CK bound to a transition-state analogue complex (CK-TSAC). Cocrystallization of the enzyme from Torpedo californica (TcCK) with ADP-Mg(2+), nitrate, and creatine yielded a homodimer, one monomer of which was liganded to a TSAC complex while the second monomer was bound to ADP-Mg(2+) alone. The structures of both monomers were determined to 2.1 A resolution. The creatine is located with the guanidino nitrogen cis to the methyl group positioned to perform in-line attack at the gamma-phosphate of ATP-Mg(2+), while the ADP-Mg(2+) is in a conformation similar to that found in the TSAC-bound structure of the homologue arginine kinase (AK). Three ligands to Mg(2+) are contributed by ADP and nitrate and three by ordered water molecules. The most striking difference between the substrate-bound and TSAC-bound structures is the movement of two loops, comprising residues 60-70 and residues 323-332. In the TSAC-bound structure, both loops move into the active site, resulting in the positioning of two hydrophobic residues (one from each loop), Ile69 and Val325, near the methyl group of creatine. This apparently provides a specificity pocket for optimal creatine binding as this interaction is missing in the AK structure. In addition, the active site of the transition-state analogue complex is completely occluded from solvent, unlike the ADP-Mg(2+)-bound monomer and the unliganded structures reported previously.

Adenosine Diphosphate↗
PubMed2002

A novel mechanism for inhibition of HIV-1 reverse transcriptase.

The human immunodeficiency virus (HIV) epidemic is an important medical problem. Although combination drug regimens have produced dramatic decreases in viral load, current therapies do not provide a cure for HIV infection. We have used structure-based design and combinatorial medicinal chemistry to identify potent and selective HIV-1 reverse transcriptase (RT) inhibitors that may work by a mechanism distinct from that of current HIV drugs. The most potent of these compounds (compound 4, 2-naphthalenesulfonic acid, 4-hydroxy-7-[[[[5-hydroxy-6-[(4-cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]carbonyl]amino]-3-[(4-cinnamylphenyl)azo], disodium salt) has an IC(50) of 90 nM for inhibition of polymerase chain extension, a K(d) of 40 nM for inhibition of DNA-RT binding, and an IC(50) of 25-100 nM for inhibition of RNaseH cleavage. The parent compound (1) was as effective against 10 nucleoside and non-nucleoside resistant HIV-1 RT mutants as it was against the wild-type enzyme. Compound 4 inhibited HIV-1 RT and murine leukemia virus (MLV) RT, but it did not inhibit T(4) DNA polymerase, T(7) DNA polymerase, or the Klenow fragment at concentrations up to 200 nM. Finally, compound 4 protected cells from HIV-1 infection at a concentration more than 40 times lower than the concentration at which it caused cellular toxicity.

Algorithms↗
PubMed2002

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies.

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.

Animals↗
PubMed2002

Defining the mandate of proteomics in the post-genomics era: workshop report.

Research in proteomics is the next step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing genomes. This will require new technologies but it is highly likely that many of these will be developed. Looking back 10 to 20 years from now, the question is: Will we have done the job wisely or wastefully? This report summarizes the presentations made at a symposium at the National Academy of Sciences on February 25, 2002.

Computational Biology↗
PubMedDate not supplied

Relating structure to mechanism in creatine kinase.

Found in all vertebrates, creatine kinase catalyzes the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine may be viewed as a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. Consequently, creatine kinase plays a significant role in energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction. First identified in 1928, the enzyme has undergone intensive investigation for over 75 years. There are four major isozymes, two cytosolic and two mitochondrial, which form dimers and octamers, respectively. Depending on the pH, the enzyme operates by a random or an ordered bimolecular mechanism, with the equilibrium lying towards phosphocreatine production. Evidence suggests that conversion of creatine to phosphocreatine occurs via the in-line transfer of a phosphoryl group from ATP. A recent X-ray structure of creatine kinase bound to a transition state analog complex confirmed many of the predictions based on kinetic, spectroscopic, and mutagenesis studies. This review summarizes and correlates the more significant mechanistic and structural studies on creatine kinase.

Animals↗
PubMedDate not supplied

Loop movement and catalysis in creatine kinase.

Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.

Adenosine Diphosphate↗