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J R Knowles

Publications and source records attributed to J R Knowles.

At least 73 records · Page 4Linked to original sources
PubMed1987

Tinkering with enzymes: what are we learning?

It is now possible, by site-directed mutagenesis of the gene, to change any amino acid residue in a protein to any other. In enzymology, application of this technique is leading to exciting new insights both into the mechanism of catalysis by particular enzymes, and into the basis of catalysis itself. The precise and often delicate changes that are being made in and near the active sites of enzymes are illuminating the interdependent roles of catalytic groups, and are allowing the first steps to be taken toward the rational alteration of enzyme specificity and reactivity.

Amino Acids↗
PubMed1987

The consequences of stepwise deletions from the signal-processing site of beta-lactamase.

Amino acids have been deleted from the processing site of pre-beta-lactamase, either into the signal sequence or into the mature protein. Whereas the loss of more than 2 amino acid residues from the C-terminal end of the signal sequence prevents the translocation of the protein into the periplasm, the removal of two or more amino acids from the beginning of the mature protein has no effect on the translocation of the truncated protein. The insertion of an additional one to three amino acids at the processing site has no detectable phenotypic consequence either. It appears that many sequences for the first few residues of the mature protein allow successful translocation and processing. In sharp contrast, the removal of one (but not both) of the amino acids that flank the processing site results in a severe growth defect in the host cell and very low expression of the protein. Yet removal of two amino acids from either side of the processing site, or removal of both the flanking residues of the processing site, results in normal secretion and signal cleavage. These results illustrate the limits on the amino acid sequence around the processing junction and suggest that interference with the signal cleavage step can lead not only to aborted secretion but also to pleiotropic consequences for the growth of the host organism.

Amino Acid Sequence↗
PubMed1987

Energetics of enzyme catalysis. I. Isotopic experiments, enzyme interconversion, and oversaturation.

An enzyme-catalyzed interconversion of one substrate, S, and one product P, by an enzyme that exists in two forms E1 and E2 where E1 binds S and E2 binds P, is considered S + E1 in equilibrium E1S in equilibrium E2P in equilibrium E2 + P. Under reversible conditions (where the concentrations of S and P are not far removed from their equilibrium values) it is shown that, in addition to the usual unsaturated and saturated behaviour there exists a third regime at high substrate concentration: the oversaturated region. In this region, the rate-limiting transition state is the interconversion of the unliganded forms of the enzyme: E1 and E2. Expressions for six different experiments involving deuterium, tritium and 14C labels are presented. By considering the results from these experiments, the nature and importance of the enzyme interconversion steps can be elucidated.

Catalysis↗
PubMed1987

Energetics of enzyme catalysis. II. Oversaturation, case diagrams, reversible and irreversible behaviour.

Most enzymes react in vivo under reversible conditions where the substrate and product concentrations are not far removed from equilibrium values. Under these conditions when the concentration of substrate is increased, in addition to the usual unsaturated and saturated behaviour we find a third type of kinetic regime at high substrate concentration-oversaturation. In this regime the rate limiting transition state involves interconversion of free enzyme forms. For a one substrate/one product enzyme, case diagrams can be constructed which depict the kinetic behaviour as a function of substrate and product concentrations. Six different cases are found and are discussed with the relevant free energy profiles. A systematic procedure is described for the investigation and construction of the case diagram.

Catalysis↗
PubMed1986

Reaction energetics of a mutant triosephosphate isomerase in which the active-site glutamate has been changed to aspartate.

The essential catalytic base at the active site of the glycolytic enzyme triosephosphate isomerase is the carboxylate group of Glu-165, which directly abstracts either the 1-pro-R proton of dihydroxyacetone phosphate or the 2-proton of (R)-glyceraldehyde 3-phosphate to yield the cis-enediol intermediate. Using the methods of site-directed mutagenesis, we have replaced Glu-165 by Asp. The three enzymes chicken isomerase from chicken muscle, wild-type chicken isomerase expressed in Escherichia coli, and mutant (Glu-165 to Asp) chicken isomerase expressed in E. coli have each been purified to homogeneity. The specific catalytic activities of the two wild-type isomerases are identical, while the specific activity of the mutant enzyme is reduced by a factor of about 1000. The observed kinetic differences do not derive from a change in mechanism in which the aspartate of the mutant enzyme acts as a general base through an intervening water molecule, because the D2O solvent isotope effects and the stoichiometries of inactivation with bromohydroxyacetone phosphate are identical for the wild-type and mutant enzymes. Using the range of isotopic experiments that were used to delineate the free-energy profile of the wild-type chicken enzyme, we here derive the complete energetics of the reaction catalyzed by the mutant protein. Comparison of the reaction energetics for the wild-type and mutant isomerases shows that only the free energies of the transition states for the two enolization steps have been seriously affected. Each of the proton abstraction steps is about 1000-fold slower in the mutant enzyme. Evidently, the excision of a methylene group from the side chain of the essential glutamate has little effect on the free energies of the intermediate states but dramatically reduces the stabilities of the transition states for the chemical steps in the catalyzed reaction.

Aspartic Acid↗
PubMed1986

Biotin-dependent carboxylation catalyzed by transcarboxylase is a stepwise process.

To investigate the mechanism of the carboxylation of pyruvate to oxalacetate catalyzed by the enzyme transcarboxylase, we have measured the D(V/K) and 13(V/K) isotope effects. Comparison of the double-reciprocal plots of the initial velocities with [1H3]pyruvate and with [2H3]pyruvate as substrate yields a deuterium isotope effect on Vmax/Km of 1.39 +/- 0.04. The 13C kinetic isotope effect on the carboxylation of pyruvate to oxalacetate has been measured by the competitive method and is 1.0227 +/- 0.0008. To determine whether the removal of the proton from pyruvate and the addition of the carboxyl group occur in the same or in different steps, the double-isotope fractionation test has been used. When [2H3]pyruvate replaces [1H3]pyruvate as the substrate, the observed 13(V/K) isotope effect falls from 1.0227 to 1.0141 +/- 0.001. The latter value is in excellent agreement with the value of 1.0136, predicted for a stepwise pathway. We may conclude, therefore, that the carboxylation of pyruvate catalyzed by transcarboxylase proceeds by a stepwise mechanism involving the intermediate formation of the substrate carbanion.

Biotin↗
PubMed1986

Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes.

The interconversion of L- and D-proline catalyzed by proline racemase has been studied. The entire time course of the approach to equilibrium has been followed. After a short time the product concentration is significant, and the reaction runs under reversible conditions. As the total substrate concentration is increased, the system moves from the unsaturated regime into the saturated regime. At very high substrate levels under the reversible conditions used, the rate constant for substrate racemization falls, as the system moves into the "oversaturated" regime. Here, the net rate of the enzyme-catalyzed reaction is limited by the rate of return of the free enzyme from the form that liberates product back to the form that binds substrate. The results are analyzed in terms of the simple mechanism (table; see text) and illustrate the additional information that is available from reactions studied under reversible conditions. In the unsaturated region the value of the second-order rate constant kU (equivalent to kcat/Km) is 9 X 10(5) M-1 s-1 in each direction. In the saturated region, kcat = kcat = 2600 s-1 and Km = 2.9 mM. In the oversaturated region, the rate constant kO is 81 M s-1. The substrate concentration at which unsaturated and saturated terms contribute equally is 2.9 mM, and the substrate concentration at which saturated and oversaturated terms contribute equally is 125 mM.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: tracer perturbation experiments using [14C]proline that measure the interconversion rate of the two forms of free enzyme.

Proline racemase exists in two states, one of which binds and isomerizes L-proline and the other of which binds and isomerizes D-proline. In the enzyme-catalyzed racemization of proline at high substrate concentrations, the interconversion of the two forms of the free enzyme becomes rate limiting. The tracer perturbation method of Britton (1966, 1973) vividly demonstrates the kinetic importance of this enzyme interconversion under oversaturating conditions and allows an estimate of the rate constant for this reaction of 10(5) s-1. It is further shown that the enzyme is bound state saturated and the peak-switch concentration, Cp, is 125 mM. At substrate concentrations higher than 125 mM the enzyme becomes oversaturated, and the reaction rate is limited by the transition state for the interconversion of two forms of the free enzyme. It seems likely that the two free enzyme forms differ only in the protonation states of the acidic and basic groups at the active site.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: transition-state fractionation factors for the two protons involved in the catalytic steps.

The isotope effects for the interconversion of L-proline and D-proline, catalyzed by proline racemase, have been determined in the saturated region with both [2-2H]proline and [2-3H]proline. The deuterium fractionation factors for each of the protons in flight have been obtained from two kinds of experiment: by measuring the rate of racemization of one [2-2H]proline enantiomer as it racemizes into an equilibrated pool of unlabeled proline and by measuring the deuterium content of a proline sample at the optical rotation maximum that occurs when an equimolar mixture of one deuterium-labeled enantiomer and the other unlabeled enantiomer runs to equilibrium. The tritium fractionation factors for each of the protons in flight have been determined from measurements of the rate of loss of tritium to the solvent as one [2-3H]proline enantiomer runs to equilibrium. Good agreement is found among the fractionation factors determined by each method. The deuterium fractionation factors for the two protons are not identical: that for the proton derived from L-proline is 0.375 and that for the proton derived from D-proline is 0.44. This difference has been confirmed by a double-competition experiment in which the optical rotation of a mixture of DL-[2-2H]proline and unlabeled DL-proline is followed with time. The rotation (initially zero) passes through a maximum, from which the ratio of the two fractionation factors (0.86) is obtained. These data, coupled with the equilibrium fractionation factor for the 2-position of proline (which has been determined to be 1.17), provide the transition-state factors for each of the in-flight protons, and delineate the nature of the transition state(s) for the enzyme-catalyzed racemization.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance.

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: fractionation factors for the essential catalytic groups in the enzyme-substrate complexes.

The fractionation factors of protons bound to the essential catalytic groups in proline racemase have been determined by comparison of the time courses of two competitive deuterium washout experiments. The rate of achievement of the maximum perturbation in the optical rotation has been measured in the oversaturated region (that is, at high substrate concentrations) under two conditions: in the first, we start with an equimolar mixture of deuterated substrate S' and of unlabeled product P; in the second, we again start with equal concentrations of substrate and product, but the concentration of the deuterated material S' is less than 20% that of S. The different concentrations of deuterated substrate produce different levels of deuteration of the enzyme's catalytic groups, the kinetic consequence of which allow the fractionation factors of these enzymic groups to be determined. The observed values for the fractionation factors of the enzyme's groups of 0.55 +/- 0.1 are only consistent with these groups' being thiols. This conclusion is supported by results of measurements of the solvent isotope effect determined in the unsaturated regime. These findings confirm the earlier suggestion of Abeles and his group that two cysteine residues mediate the catalysis of proline racemization by this enzyme.

Amino Acid Isomerases↗
PubMed1986

Energetics of proline racemase: rates, fractionation factors, and buffer catalysis in the oversaturated region. Nature of the interconversion of the two forms of free enzyme.

To probe the nature of the interconversion of the two unliganded forms of proline racemase, a number of experiments have been performed under oversaturating conditions where the rate of the enzymic reaction is mainly limited by the rate of this interconversion. Competitive deuterium washout experiments, where an equimolar mixture of D- and L-proline (in which some or all of one enantiomer is specifically deuterated at the 2-position) is allowed to reach chemical and isotopic equilibrium mediated by the enzyme, have been followed in four ways. The size and the rate of achievement of the maximum perturbation in the optical rotation have been measured, the deuterium content of the substrate at this maximum has been determined, and the final approach to equilibrium after the perturbation maximum has been followed. Further, the enzyme-catalyzed rate of tritium loss from [2-3H]proline has been established. Finally, it has been shown that the enzyme interconversion reaction is catalyzed by several buffers (such as ammonium, hydrazinium, and hydrogen sulfide). These data are discussed in terms of Marcus' theory, which allows a rather detailed picture of the mechanism of free enzyme interconversion to be drawn. This process nicely parallels the mechanism of the enzyme-catalyzed interconversion of the proline enantiomers, and it is evident that substrate racemization (with the concomitant switch of the enzyme-bound protons) is mirrored by the water-mediated switch of the enzyme-bound protons that effects the interconversion of the free enzyme forms. The results favor a stepwise reaction for the interconversion of the free enzyme forms in which a proton is abstracted from a bound water molecule to give a reaction intermediate having a hydroxide ion bound to the diprotonated form of the enzyme.

Amino Acid Isomerases↗
PubMed1986

Secretion of beta-lactamase into the periplasm of Escherichia coli: evidence for a distinct release step associated with a conformational change.

The secretion of beta-lactamase (EC 3.5.2.6) into the periplasm of Escherichia coli has been followed by pulse-chase labeling at 15 degrees C. Though the periplasmic fraction contains only the mature form of the enzyme, the spheroplast fraction contains the completed precursor and a hitherto undocumented processed form. When whole spheroplasts are treated with trypsin, the processed form in this fraction is completely digested. This is in contrast to the native mature enzyme localized in the periplasm, which is trypsin resistant. The beta-lactamase is evidently processed after translocation to a trypsin-sensitive form that is transiently bound to the periplasmic face of the inner membrane. The release of this processed form into the periplasm occurs concomitantly with a conformational change that results in the soluble, catalytically active, trypsin-resistant structure.

Cell Compartmentation↗
PubMed1985

Signal sequence mutants of beta-lactamase.

The function of the NH2-terminal signal peptide in the translocation of beta-lactamase across the inner membrane of Escherichia coli has been studied by characterization of 15 signal sequence mutants. Three amino acid substitutions (Pro 20 to Ser, Pro 20 to Phe, and Cys 18 to Tyr) in the 23-amino acid signal sequence each cause, to varying degrees, a defect in the proteolytic processing of pre-beta-lactamase, abnormal growth of the host strain, and a severe reduction in the expression of beta-lactamase in vivo but not in vitro. The results are consistent with a model for protein secretion in E. coli that parallels the pathway proposed for translocation across the endoplasmic reticulum in eucaryotic cells.

Alleles↗
PubMed1985

A simple and efficient method for chemical mutagenesis of DNA.

A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA segment is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragment is obtained. This methoxylamine-derivatized DNA segment is then joined with linearized M13 RF DNA, competent E. coli is transfected, and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM beta-lactamase.

Base Sequence↗