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C D Poulter

Publications and source records attributed to C D Poulter.

At least 37 records · Page 2Linked to original sources
PubMed2000

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

Amino Acid Sequence↗
PubMed1999

Synthesis of (R)-[2-2H]isopentenyl diphosphate and determination of its enantiopurity by 2H NMR spectroscopy in a lyotropic medium.

[formula: see text] The synthesis of (R)-[2-2H]isopentenyl diphosphate from D-mannitol 1,2:5,6-bis-acetonide in 10 steps is reported. Stereospecific incorporation of the label is achieved by a BF3-catalyzed NaCNBD3 reduction of the enantiomerically pure (S)-isopropylidene oxirane intermediate. The enantiomeric excess of the penultimate precursor [2-2H]isopentenyl tosylate (> 95% ee) was determined by 2H NMR spectroscopy in a poly-gamma-benzyl-L-glutamate/CH2Cl2 liquid crystal at -50 degrees C.

Hemiterpenes↗
PubMed1999

Yeast protein farnesyltransferase. pKas of peptide substrates bound as zinc thiolates.

Protein farnesyltransferase (PFTase) is a zinc metalloenzyme that catalyzes the posttranslational alkylation of the cysteine in C-terminal -Ca(1)a(2)X sequences by a 15-carbon farnesyl residue, where C is cysteine, a(1) and a(2) are normally aliphatic amino acids, and X is an amino acid that specifies selectivity for the farnesyl moiety. Formation of a Zn(2+) thiolate in the PFTase. peptide complex was detected by the appearance of an absorbance at 236 nm (epsilon = 15 000 M(-1) cm(-1)), which was dependent on the concentration of peptide, in a UV difference spectrum in a solution of PFTase and the peptide substrate RTRCVIA. We developed a fluorescence anisotropy binding assay to measure the dissociation constants as a function of pH for peptide analogues by appending a 2',7'-difluorofluorescein to their N-terminus. The electron-withdrawing fluorine atoms allowed us to measure peptide binding down to pH 5.5 without having to correct for the changes in fluorescence intensity that accompany protonation of the fluorophore. Measurements of the pK(a)s for thiol groups in free and bound peptide indicate that peptide binding is accompanied by formation of a zinc thiolate and that binding to PFTase lowers the pK of the peptide thiol by 3 units. In similar studies with the betaY310F mutant, the pK(a) of the thiol moiety was lowered by 2 units upon binding, indicating that the hydroxyl group in the conserved tyrosine helps stabilize the bound thiolate.

Alkyl and Aryl Transferases↗
PubMed1999

Yeast protein farnesyltransferase. Binding of S-alkyl peptides and related analogues.

[reaction: see text] Protein farnesyltransferase (PFTase) catalyzes alkylation of cysteine residues by farnesyl diphosphate (FPP). The dissociation constants for the PFTase-peptide analogue complexes for the series of analogues fl-RTRC(X)VIA (X = H, methyl, dodecyl, farnesyl) were measured by fluorescence anisotropy. The results indicate that an ionizable sulfhydryl moiety is important for substrate binding and the farnesyl group in the product facilitates binding.

Alkyl and Aryl Transferases↗
PubMed1999

Farnesyl protein transferase: identification of K164 alpha and Y300 beta as catalytic residues by mutagenesis and kinetic studies.

Farnesyl protein transferase (FPT) is an alpha/beta heterodimeric zinc enzyme that catalyzes posttranslational farnesylation of many key cellular regulatory proteins, including oncogenic Ras. On the basis of the recently reported crystal structure of FPT complexed with a CVIM peptide and alpha-hydroxyfarnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding and catalysis could be identified. Eight single mutants, including K164N alpha, Y166F alpha, Y166A alpha, Y200F alpha, H201A alpha, H248A beta, Y300F beta, and Y361F beta, and a double mutant, H248A beta/Y300F beta, were prepared. Steady-state kinetic analysis along with structural evidence indicated that residues Y200 alpha, H201 alpha, H248 beta, and Y361 beta are mainly involved in substrate binding. In addition, biochemical results confirm structural observations which show that residue Y166 alpha plays a key role in stabilizing the active site conformation of several FPT residues through cation-pi interactions. Two mutants, K164N alpha and Y300F beta, have moderately decreased catalytic constants (kcat). Pre-steady-state kinetic analysis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41- and 30-fold, respectively. The product-releasing rate for each dropped approximately 10-fold. In pH-dependent kinetic studies, Y300F beta was observed to have both acidic and basic pKa values shifted 1 log unit from those of the wild-type enzyme, consistent with a possible role for Y300 beta as an acid-base catalyst. K164N alpha had a pKa shift from 6.0 to 5.3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a possible FPT reaction mechanism is proposed with both Y300 beta and K164 alpha playing key catalytic roles in enhancing the reactivity of the farnesyl diphosphate leaving group.

Alkyl and Aryl Transferases↗
PubMed1999

Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase.

Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s. The E. coli protein requires Mg(2+) or Mn(2+) for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.

Carbon-Carbon Double Bond Isomerases↗
PubMed1998

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana.

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.

Amino Acid Sequence↗
PubMed1997

Yeast protein farnesyltransferase. Site-directed mutagenesis of conserved residues in the beta-subunit.

Protein prenyltransferases catalyze the posttranslational modification of cysteines by isoprenoid hydrocarbon chains. A protein farnesyltransferase (PFTase) and a protein geranylgeranyltransferase (PGGTase-I) alkylate cysteines in a CaaX C-terminal tetrapeptide sequence, where a is usually an aliphatic amino acid and X is an amino acid that specifies whether a C15 farnesyl or C20 geranylgeranyl moiety is added. A third enzyme, PGGTase-II, adds geranylgeranyl groups to both cysteines at the C-terminus of Rab proteins. All three enzymes are Zn2+ metalloproteins and also require Mg2+ for activity. The protein prenyltransferases are heterodimers. PFTase and PGGTase I contain identical alpha-subunits and distinctive beta-subunits, which are responsible for the differences in substrate selectivity seen for the two enzymes. The subunits in PGGTase-II are similar, but not identical, to their counterparts in the other two enzymes. An alignment of amino acid sequences for the beta-subunits of all three enzymes shows five regions of high similarity. Thirteen of the conserved polar and charged residues in yeast PFTase were selected for substitution by site-directed mutagenesis. Kinetic studies revealed a subset of five enzymes, R211Q, D307A, C309A, Y310F, and H363A, with substantially reduced catalytic constants (kcat). Metal analyses of wild-type enzyme and the five least reactive mutants showed that the substitutions had compromised Zn2+ binding in the D307A, C309A, and H363A enzymes.

Alkyl and Aryl Transferases↗
PubMed1997

Pre-steady-state study of recombinant sesquiterpene cyclases.

An Escherichia coli expression system was used to generate hexahistidyl-tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step. Genes for Hyoscyamus muticus vetispiradiene synthase (HVS), a chimeric 5-epi-aristolochene synthase (CH3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (CH4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 50% of the total protein and 35% of the soluble protein. From initial velocity experiments, the Michaelis constant for HVS was 3.5 microM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 microM, respectively. Steady-state catalytic constants were from 0.02 to 0.04 s-1. A combination of pre-steady-state rapid quench experiments, isotope trapping experiments, and experiments to measure the burst rate constant as a function of substrate concentration revealed that turnover in all three cyclases is limited by a step after the initial chemical step involving rupture of the carbon-oxygen bond in farnesyl diphosphate (FPP). Rate constants for the limiting step were 10-70-fold smaller than for the initial chemical step. Dissociation constants for the enzyme-substrate complex (20-70 microM) were determined from the pre-steady-state experiments and were significantly larger than the observed Michaelis constants. A mechanism that involves an initial, rapid equilibration of enzyme with substrate to form an enzyme-substrate complex, followed by a slower conversion of FPP to an enzyme-bound hydrocarbon and a subsequent rate-limiting step, is proposed for the three enzymes. Interestingly, the multifunctional chimeric enzyme CH4 exhibited both a tighter binding of FPP and a faster conversion of FPP to products than either of its wild-type parents.

Alkyl and Aryl Transferases↗
PubMed1997

Yeast protein farnesyltransferase: a pre-steady-state kinetic analysis.

Protein farnesyltransferase catalyzes alkylation of the cysteine in a carboxy-terminal CaaX motif where a is typically an aliphatic amino acid and X is alanine, methionine, serine, glutamine, or cysteine by a farnesyl residue. The modification enhances the lipophilicity of farnesylated proteins and promotes their association with membranes as part of their normal cellular function. Among the proteins modified by farnesyl residues is Ras, an important component in the signal transduction network for cell division that has been implicated in several forms of human cancer. In this paper, we describe isotope trapping, rapid quench, and single turnover experiments with the yeast enzyme using farnesyl diphosphate and the short peptide RTRCVIA as substrates. The kinetic constants for substrate binding, chemistry, and product release were determined from a fit of the differential equations describing the minimal catalytic mechanism to the kinetic data by numerical integration. Rate constants for chemistry and product release were 10.5 and 3.5 s(-1), respectively. The dissociation rate constant (33 s(-1)) for release of peptide from the ternary enzyme-substrate complex was three times larger than the rate constant for chemistry. The enthalpy of reaction, delta Hrxn = -17 +/- 1 kcal/mol for farnesylation of cysteine, was measured by microcalorimetry. Isotope trapping experiments revealed that the enzyme-farnesyl diphosphate complex was efficiently trapped by peptide but that the enzyme-peptide complex was not trapped by farnesyl diphosphate. These results are consistant with an ordered mechanism for formation of a catalytically competent ternary enzyme-farnesyl diphosphate-peptide complex.

Alkyl and Aryl Transferases↗
PubMed1997

Yeast protein geranylgeranyltransferase type-I: steady-state kinetics and substrate binding.

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in Leu or Phe by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). A kinetic study of the alkylation reaction was conducted with a continuous assay based on the fluorescence enhancement that accompanies geranylgeranylation of dansyl-GCIIL. The kinetic constants k(cat) = 0.34 +/- 0.01 s(-1), K(M)(G) = 0.86 +/- 0.05 microM for GGPP, and K(M)(D) = 1.6 +/- 0.1 microM for dansyl-GCIIL were calculated from initial rates measured at varying concentrations of the substrates. Inhibitor studies were conducted with dead-end inhibitors for GGPP and the peptide substrate. Double reciprocal plots for the peptide mimic Cys-AMBA-Leu gave a competitive pattern when plotted against varying concentrations of dansyl-GCIIL and an uncompetitive pattern against GGPP. Similar plots for 1-phosphono-(E,E,E)-geranylgeraniol, a dead-end inhibitor for GGPP, gave a competitive double reciprocal plot for varied concentrations of GGPP and induced potent substrate inhibition by dansyl-GCIIL when dansyl-GCIIL was the varied substrate. The dissociation constant (K(D)) for the PGGTase-I x GGPP complex was 120 +/- 20 nM. These results are consistent with an ordered binding mechanism for PGGTase-I where GGPP adds before peptide.

Alkyl and Aryl Transferases↗
PubMed1997

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: a binding mechanism for recombinant enzyme.

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The enzyme, encoded by the miaA gene, was overproduced and purified to apparent homogeneity in three steps by ion-exchange (DE52 and Mono-Q) and size exclusion chromatography. Affinity-tagged DMAPP-tRNA transferase containing a C-terminal tripeptide alpha-tubulin epitope also was overproduced and purified to apparent homogeneity in two steps by ion-exchange and immunoaffinity chromatography. Addition of the C-terminal tripeptide alpha-tubulin epitope to DMAPP-tRNA transferase did not affect the activity of the enzyme. Undermodified tRNA(Phe) used as substrate in the DMAPP-tRNA transferase-catalyzed reaction was isolated and purified from an overexpressing clone in a miaA deficient strain of E. coli. Active recombinant E. coli DMAPP-tRNA transferase is monomeric. The enzyme transferred the dimethylallyl moiety of DMAPP to A37, located adjacent to the anticodon in undermodified tRNA(Phe). The enzyme required Mg2+ for activity and exhibited a broad pH optimum. Michaelis constants for tRNA(Phe) and DMAPP are 96 +/- 11 nM and 3.2 +/- 0.5 microM, respectively, and Vmax = 0.83 +/- 0.02 micromol min-1 mg-1. DMAPP-tRNA transferase bound tRNA(Phe) with a dissociation constant of 5.2 +/- 1.2 nM. In contrast, DMAPP did not bind to the enzyme in the absence of tRNA. However, DMAPP was bound with a dissociation constant of 3.4 +/- 0.6 microM in the presence of a minihelix analogue of the anticodon stem-loop of tRNA(Phe) where the base corresponding to A37 was replaced by inosine. These results suggest an ordered sequential mechanism for substrate binding.

Alkyl and Aryl Transferases↗
PubMed1997

Chain elongation in the isoprenoid biosynthetic pathway.

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.

Alkyl and Aryl Transferases↗
PubMed1996

Regulation of product chain length by isoprenyl diphosphate synthases.

An analysis of the x-ray structure of homodimeric avian farnesyl diphosphate synthase (geranyltransferase, EC 2.5.1.10) coupled with information about conserved amino acids obtained from a sequence alignment of 35 isoprenyl diphosphate synthases that synthesize farnesyl (C15), geranylgeranyl (C20), and higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for determining the ultimate length of the hydrocarbon chains. This hypothesis was supported by site-directed mutagenesis to transform wild-type avian farnesyl diphosphate synthase (FPS) into synthases capable of producing geranylgeranyl diphosphate (F112A), geranylfarnesyl (C25) diphosphate (F113S), and longer chain prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure of the F112A/F113S mutant in the apo state and with allylic substrates bound produced the strongest evidence that these mutations caused the observed change in product specificity by directly altering the size of the binding pocket for the growing isoprenoid chain in the active site of the enzyme. The proposed binding pocket in the apo mutant structure was increased in depth by 5.8 A as compared with that for the wild-type enzyme. Allylic diphosphates were observed in the holo structures, bound through magnesium ions to the aspartates of the first of two conserved aspartate-rich sequences (D117-D121), with the hydrocarbon tails of all the ligands growing down the hydrophobic pocket toward the mutation site. A model was constructed to show how the growth of a long chain prenyl product may proceed by creation of a hydrophobic passageway from the FPS active site to the outside surface of the enzyme.

Alkyl and Aryl Transferases↗
PubMed1996

Yeast geranylgeranyltransferase type-II: steady state kinetic studies of the recombinant enzyme.

Rab proteins in mammalian cells, or Ypt1p and Sec4p in yeast, regulate vesicular traffic. Prenylation of these small GTP-binding proteins is required for membrane attachment and subsequent biological activity. Yeast protein geranylgeranyltransferase type-II (PGGTase-II) catalyzes the prenylation of Ypt1p in the presence of an escort protein, Msi4p. The genes encoding the alpha-(BET4) and beta-(BET2) subunits of PGGTase-II were translationally coupled by overlapping the BET4-BET2 stop/start codons and by adding a ribosome-binding site near the 3'-end of BET4 that fused an -EEF C-terminal alpha-tubulin epitope to Bet4p. Active recombinant heterodimer was purified by chromatography on DE52 and anti-alpha-tubulin columns. Recombinant Msi4p with an N-terminal polyhistidine leader was purified on a Ni(2+)-Sepharose column, followed by gel filtration and ion exchange chromatography. An escort protein, Msi4p, was necessary for geranylgeranylation of Ypt1p by yeast PGGTase-II. Michaelis constants for GGPP and Ypt1p were 1.6 and 1.1 microM, respectively; Vmax = 1.7 nmol min-1 mg-1 for yeast PGGTase-II. Typical Michaelis-Menten behavior was also seen for the enzyme for varied concentrations of Msi4p, with a maximal catalytic activity seen for a 10-fold excess of escort protein over enzyme. In contrast to previous reports, PGGTase-II requires both Zn2+ and Mg2+ for maximal activity, although Zn2+ becomes inhibitory at concentrations above approximately 10 microM. Prenylated Ypt1p obtained after incubation of Ypt1p with PGGTase-II, Msi4p, and geranylgeranyl diphosphate was digested with trypsin. The C-terminal peptide fragment from modified Ypt1p was purified by HPLC and analyzed by electrospray mass spectrometry. The mass of the fragment is consistent with the 12-mer C-terminal amino acid fragment predicted from proteolysis by trypsin with both cysteine residues modified by geranylgeranyl moieties.

Alkyl and Aryl Transferases↗
PubMed1996

Human isopentenyl diphosphate: dimethylallyl diphosphate isomerase: overproduction, purification, and characterization.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A human cDNA sequence [J. Xuan, J. Kowalski, A.F. Chambers, and D.T. Denhardt (1994) Genomics 20, 129-131] containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases [F.M. Hahn and C.D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The human cDNA sequence was cloned into expression plasmid pFMH12. The encoded protein was overproduced in Escherichia coli and purified to > 90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography. The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+. The Michaelis constant for IPP was 33 microM, similar to the value of 43 microM reported for yeast IPP isomerase; Vmax = 4.1 mumol min-1 mg-1 for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme [I.P. Street and C.D. Poulter (1990) Biochemistry 29, 7531-7538].

Amino Acid Sequence↗