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J R Knowles

Publications and source records attributed to J R Knowles.

At least 55 records · Page 3Linked to original sources
PubMed1989

Evolutionary optimization of the catalytic effectiveness of an enzyme.

The kinetic and thermodynamic features of reactions catalyzed by present-day enzymes appear to be the consequence of the evolution of these proteins toward maximal catalytic effectiveness. These features are identified and analyzed (in detail for one substrate-one product enzymes) by using ideas that link the energetics of the reaction catalyzed by an enzyme to the maximization of its catalytic efficiency. A catalytically optimized enzyme will have a value for the "internal" equilibrium constant (Kint, the equilibrium constant between the substrates and the products of the enzyme when all are bound productively) that depends on how close to equilibrium the enzyme maintains its reaction in vivo. Two classes are apparent. For an enzyme that operates near equilibrium, the catalytic efficiency is sensitive to the value of Kint, and the optimum value of Kint is near unity. For an enzyme that operates far from equilibrium, the catalytic efficiency is less sensitive to the value of Kint, and Kint assumes a value that ensures that the rate of the chemical transformation is equal to the rate of product release. In each of these cases, the internal thermodynamics is "dynamically matched", where the concentrations of substrate- and product-containing complexes are equal at the steady state in vivo.

Biological Evolution↗
PubMed1989

Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase.

The equilibrium constant (Kint) for the enzyme-bound substrate and product of a one substrate/one product enzyme (enolase) and for those of a two substrate/two product enzyme (creatine kinase) have been determined. The values of Kint were determined by the rapid quenching of equilibrium mixtures of enzyme and radiolabeled substrate and product, under conditions where all of the marker substrate and product are bound. The scope and limitations of this method are discussed. Values of Kint have been collected from the literature, and it is shown that these data are consistent with the theory for kinetically optimized enzymes that is developed in the preceding paper.

Adenosine Diphosphate↗
PubMed1989

A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in Escherichia coli. Implications for the mechanism of protein secretion.

A hybrid protein that comprises the beta-lactamase signal peptide fused precisely to chicken muscle triosephosphate isomerase is not secreted into the periplasm of Escherichia coli. The protein can be secreted, however, if an arginine residue at position 3 of the isomerase is replaced by either a serine or a proline residue. In contrast, replacement of a neighboring lysine residue has no effect on secretion of the protein. Furthermore, if the arginine is removed from position 3 to generate a secreted protein, but is then reintroduced in place of the neighboring lysine, the blockade to secretion is re-established. The singular effect of the arginine residue on secretion does not result from the role this residue plays in the formation or stabilization of the native isomerase structure: mutational alterations remote from the N terminus of the isomerase that prevent the proper folding of the protein do not relieve the block to secretion. The finding that an arginine residue prevents secretion while a lysine residue does not, suggests that basic residues near the mature N terminus of a secreted protein must be deprotonated if orderly export is to occur. This implies that the signal peptide along with the N-terminal portion of the mature protein partitions directly into the lipid bilayer in the course of the secretory process.

Amino Acid Sequence↗
PubMed1989

Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein.

The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli. The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed. At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein. The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase. It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R. (1989) J. Biol. Chem. 264, 20082-20088). With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid. The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E. coli.

Amino Acid Sequence↗
PubMed1989

Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis.

The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.

Apoenzymes↗
PubMed1989

Dehydroquinate synthase: the use of substrate analogues to probe the early steps of the catalyzed reaction.

The early steps of the proposed mechanistic pathway for dehydroquinate synthase have been probed with a series of substrate analogues. These analogues, 3-9, are structurally prohibited from undergoing the beta-elimination of inorganic phosphate that represents the committed step in the conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). In agreement with previous observations, the analogues that possess shortened side chains (3,5, and 6) bind more tightly to the enzyme than those (4 and 7-9) that are more nearly isosteric with the substrate. Two hitherto unrecognized factors that influence binding have been identified: (i) carbacylic analogues bind 25-100 times more tightly than the corresponding oxacyclic materials (indeed, the carbacyclic phosphonate 5 has a Ki value of 8 x 10(-10)M) and (ii) the side chain appears to be bound in a gauche conformation similar to the most stable conformation of the cis-vinylhomophosphonate 8. These trends in binding can be rationalized by considering the behavior of the analogues in the first two chemical steps of the mechanism: NAD+-mediated oxidation at C-5 and enolization at C-6 (the first part of the E1cB elimination of inorganic phosphate). Direct spectrophotometric determination of the equilibrium level of enzyme-bound NADH indicates that the carbacyclic analogues are more readily oxidized than the oxacyclic compounds, and this predictable difference in redox behavior is reflected in the observed differences in binding. The gauche conformation of the C-7 side chain appears to be required for proton abstraction from C-6, since only those analogues that can adopt this conformation undergo enzyme-catalyzed exchange of the C-6 proton with the solvent. This conformation positions one of the peripheral oxygens of the phosphate (or phosphonate) group close to the C-6 proton. Taken together with other data, these results suggest that the enzyme exploits this substrate base in the enolization, which occurs through an intramolecular proton transfer. The loss of Pi then completes the beta-elimination.

Catalysis↗
PubMed1989

Dehydroquinate synthase: the use of substrate analogues to probe the late steps of the catalyzed reaction.

The later steps of the proposed mechanistic pathway for the reaction catalyzed by dehydroquinate synthase have been probed by using three substrate analogues. Each of these analogues is structurally prohibited from undergoing the ring-opening reaction that necessarily precedes the carbon-carbon bond-forming step in the overall conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). Two of the analogues (the 2-deoxy cyclic compound 3 and the carbacyclic material 4) are locked into a cyclic form, mimicking the pyranose form of the substrate DAHP. The third analogue, 5, contains no carbonyl group at C-2 and may thus resemble the open-chain form of DAHP. Analogues 3 and 4 each bind to the enzyme and are competitive inhibitors having Ki values of 35 and 0.12 microM, respectively. More importantly, however, incubation of these analogues with the enzyme leads to the catalytic production of Pi along with the corresponding exomethylene compounds that are analogous to the enol ether IV postulated for the normal synthase reaction. In contrast to these results, the acyclic analogue 5 is neither a substrate nor an inhibitor of the enzyme. These data suggest that the enzyme recognizes and acts upon the alpha-pyranose form of the natural substrate. The ready release of the exomethylene products from the processing of analogues 3 and 4 is consistent with the suggestion of Bartlett and his group that the enzyme may release the enol ether intermediate IV into solution, where the ring opening and cyclization occur nonenzymically. The use of 3 stereospecifically labeled with deuterium at C-7 allows the sterochemical course of the beta-elimination of phosphate to be established. This step proceeds with syn stereochemistry, which fits the pattern of enzyme-catalyzed elimination from substrates where the proton is lost from a position alpha to a ketone, an aldehyde, or a thiolester. Since the overall stereochemical course of the transformation mediated by dehydroquinate synthase had been shown to be inversion, the present finding of a syn elimination suggests that the transition state for the subsequent intramolecular aldol reaction has a chairlike geometry.

Catalysis↗
PubMed1989

Is the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive), a copper metalloenzyme?

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive) was purified from Escherichia coli carrying the plasmid pKB45. Enzyme of high specific catalytic activity (70 mu/mg) was obtained from cells grown only to the early log phase. The purified protein contained Cu(II) and showed an absorption band at 350 nm. Metal-free, catalytically inactive apoenzyme could be produced by dialysis against cyanide ion, and the holoenzyme could be reconstituted in terms of both catalytic activity and A350 by the binding of one Cu(II) ion per enzyme subunit. Zn(II) also reactivated the apoenzyme to about 50% of the level seen with Cu(II), although in this case no band appeared at 350 nm. In contrast to earlier reports that the enzyme contains substoichiometric levels of iron, insignificant amounts of iron were found in the isolated enzyme, and neither Fe(II) nor FE(III) regenerated either an absorption band at 350 nm or any catalytic activity from the apoenzyme. The evident preference of the enzyme as isolated for (Cu)II suggests that the synthase might naturally be a copper metalloenzyme.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1988

The stereochemical course of phospho group transfer catalyzed by rat liver 6-phosphofructo-2-kinase.

Adenosine [gamma-(S)-16O,17O,18O]triphosphate was used as substrate in the phosphokinase reaction catalyzed by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The product D-fructose 2,6-[2-16O,17O,18O]bisphosphate was then used as substrate in the alkaline phosphatase-mediated transfer of the phospho groups to (S)-butane-1,3-diol, where the configuration at phosphorus has been determined. Although there was approximately equal transfer by alkaline phosphatase of the labeled 2- and the unlabeled 6-phospho groups, the subsequent assignment of configuration of the chiral phospho group from the 2-position was unambiguous. It was found that 6-phosphofructo-2-kinase proceeds by a pathway that results in net inversion of the configuration at phosphorus. The simplest interpretation of this result is that the phospho group is transferred directly between substrates in a ternary complex by an "in-line" mechanism not involving a phosphoenzyme intermediate. This conclusion is consistent with the fact that the enzyme cannot be labeled by [gamma-32P]ATP and with steady-state kinetic data that suggest an ordered sequential mechanism. This finding also indicates that the adenine nucleotide and sugar phosphate isotope exchange reactions catalyzed by the enzyme are not relevant to the normal catalytic pathway of the kinase.

Adenosine Triphosphate↗
PubMed1988

On the intermediacy of carboxyphosphate in biotin-dependent carboxylations.

In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). We have resorted to three mechanistic tests using the biotin carboxylase subunit of acetyl-CoA carboxylase from Escherichia coli: positional isotope exchange, intermediate trapping, and 18O tracer experiments on the ATPase activity. First, no catalysis of positional isotope exchange in adenosine 5'-[( alpha, beta-18O, beta, beta-18O2]triphosphate) was observed when either biotin or bicarbonate was absent, nor was any exchange seen in the presence of both N-1-methylbiotin and bicarbonate. Second, the putative carboxyphosphate intermediate could not be trapped as its trimethyl ester, under conditions of incubation and analysis where the authentic triester was shown to be adequately stable. In the third test, however, we showed that the ATPase activity of biotin carboxylase that is seen in the absence of biotin, an activity that is known to parallel the normal carboxylase reaction when biotin is present, occurs with the transfer of an 18O label directly from [18O]bicarbonate into the product Pi. This result suggests that the bicarbonate-dependent biotin-independent ATPase reaction catalyzed by biotin carboxylase goes via carboxyphosphate and that the carboxylation of biotin itself may proceed analogously.

Adenosine Triphosphatases↗
PubMed1988

Phosphonate biosynthesis: isolation of the enzyme responsible for the formation of a carbon-phosphorus bond.

The first isolation of a naturally occurring phosphonate in 1959 led rapidly to the discovery of a variety of metabolites containing a phosphorus-carbon bond. Phosphonates have been found in bacteria, fungi, and higher organisms such as the snail schistosome vector Biomphalaria. The biosynthetic path to the P-C bond has, however, remained undefined. Thus although it was shown twenty years ago that the isotope label from [14C]glucose or from [32P]phosphoenolpyruvate is incorporated into 2-aminoethylphosphonate by the protozoan Tetrahymena pyriformis, the presumed stoichiometric transformation of phosphoenolpyruvate to phosphonopyruvate has never been demonstrated. Low conversions of phosphoenolpyruvate into 2-aminoethylphosphonate and the trapping of phosphonopyruvate from phosphoenolpyruvate have been reported, but these reactions have not proved reproducible, and the existence of the critical enzyme, phosphoenolpyruvate phosphonomutase, has remained notional. We now report experiments that resolve this enigma, and describe the isolation and characterization of the pure mutase from T. pyriformis.

Amino Acid Sequence↗
PubMed1988

Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli.

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)-3H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the "exchange-conversion" experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism.

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.

Binding Sites↗
PubMed1988

Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test.

Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect D(V/K) of 1.3 +/- 0.1 has been found when [2H3]acetyl-CoA is the substrate. The 13C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product (containing the carbon of interest at C-2) was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO2 for isotope ratio mass spectrometric analysis. The 13C isotope effect with [1H3]acetyl-CoA [that is, 13(V/K)H] is 1.0037 +/- 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of 13(V/K)H, the value of the 13C isotope effect when deuteriated [2H3]acetyl-CoA is the substrate [that is, 13(V/K)D] can be predicted for three possible mechanisms. If 13(V/K)H is a kinetic isotope effect and the reaction is concerted, the value of the 13C effect on deuteriation of acetyl-CoA will rise to 1.011; if 13(V/K)H is a kinetic isotope effect and the reaction is stepwise, the value of the 13C effect will fall to 1.0025; and if the 13C effect is an equilibrium isotope effect deriving from glyoxylate dehydration, the reaction is necessarily stepwise, and the value of 13(V/K)D will be 1.0037, unchanged from that of 13(V/K)H. Experimentally, the value of 13(V/K)D is 1.0037 +/- 0.0007, which requires that malate synthase follow a stepwise path. It is therefore clear that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic.

Acetyl Coenzyme A↗
PubMed1988

Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase.

The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (Rp)-[16O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1988

Triosephosphate isomerase catalysis is diffusion controlled. Appendix: Analysis of triose phosphate equilibria in aqueous solution by 31P NMR.

The rates of the forward and reverse reactions of triosephosphate isomerase catalyzed by the wild-type and by a sluggish mutant enzyme have been studied in the absence and the presence of several viscosogenic agents. For the mutant enzyme, the kcat for which is some 10(3) times less than that for the wild-type enzyme, the value of kcat/Km with glyceraldehyde phosphate as substrate is almost unaffected by the presence of sucrose or glycerol, even though the concentration of the aldehyde form of the substrate is smaller because of hemiacetal formation. [The nature and relative amounts of the various forms of triose phosphate present in solution (free carbonyl forms, hydrates, dimers, hemiacetal adducts) have been evaluated by 31P NMR and are presented in the Appendix.] The viscosogenic agents cause the substrate to bind more tightly to the enzyme, roughly compensating for the lower substrate concentration. With dihydroxyacetone phosphate as substrate, the values of kcat/Km for the mutant enzyme increase with the addition of viscosogenic agent, consistent with the tighter binding of substrate without (in this case) any concomitant loss due to hemiketal formation. These results for the mutant enzyme (known to be limited in rate by an enolization step in the catalytic mechanism) can be used to interpret the behavior of the wild-type enzyme. Plots of the relative values of kcat/Km for catalysis by the wild-type enzyme (normalized with the corresponding data for the mutant enzyme) against the relative viscosity have slopes close to unity, as predicted by the Stokes-Einstein equation for a cleanly diffusive process. In the presence of polymeric viscosogenic additives such as poly(ethylene glycol), polyacrylamide, or ficoll, no effect on kcat/Km is seen for the wild-type enzyme, consistent with the expectation that molecular diffusion rates are unaffected by the macroviscosity and are only slowed by the presence of smaller agents that raise the microviscosity. These results show that the reaction catalyzed by the wild-type triosephosphate isomerase is limited by the rate at which glyceraldehyde phosphate encounters, or departs from, the active site.

Animals↗
PubMed1987

Enzyme relaxation in the reaction catalyzed by triosephosphate isomerase: detection and kinetic characterization of two unliganded forms of the enzyme.

Triosephosphate isomerase has been shown to exist in two unliganded forms, one of which binds and isomerizes (R)-glyceraldehyde 3-phosphate and the other of which binds and isomerizes dihydroxyacetone 3-phosphate. The tracer perturbation method of Britton demonstrates the kinetic significance of the interconversion of these two enzyme forms at high substrate concentrations and yields a rate constant of about 10(6) s-1 for the interconversion. Although the molecular nature of the two forms of unliganded enzyme is not defined by these experiments, a shuffling of protons among active site residues, or a protein conformational change, or both, may be involved. This study, coupled with the known rate constants for the substrate-handling steps of triosephosphate isomerase catalysis, completes the kinetic characterization of the catalytic cycle for this enzyme.

Animals↗