Steady-state Enzyme Kinetics Research Articles

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.

Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3β-Hydroxysteroid Dehydrogenase

Androgen biosynthesis requires 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) and the 17alpha-hydroxylase and 17,20-lyase activities of cytochrome P450c17. Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome. However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis. To investigate potential actions of these drugs on P450c17 and 3betaHSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments. The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3betaHSDII (K(I) = 25.4 +/- 5.1 microm) and both activities of P450c17 (K(I) for 17alpha-hydroxylase, 8.4 +/- 0.6 microm; K(I) for 17,20-lyase, 5.3 +/- 0.7 microm). The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3betaHSDII. The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3betaHSDII. These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions. Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.

23] Phosphoribosylanthranilate isomerase and indoleglycerol-phosphate synthase: Tryptophan biosynthetic enzymes from Thermotoga maritima

Publisher Summary Phosphoribosylanthranilate isomerase (PRAI) and indoleglycerol-phosphate synthase (IGPS) catalyze the fourth and the fifth steps of tryptophan biosynthesis—namely, an Amadori rearrangement of a ribosylamine and the ring closure to the indole nucleus. In Escherichia coli , PRAI and IGPS are fused to give the monomeric bifunctional polypeptide elGPS-PRAI. The artificially separated monofunctional PRAI and IGPS domains were subsequently produced and characterized, and the reaction mechanisms and folding properties of both enzymes and the PRAI from yeast (yPRAI) were studied, using stopped-flow techniques as well as site-directed mutagenesis. However, many mutational studies, especially on eIGPS, were hampered considerably by the instability and insolubility of the produced mutants. As an alternative source of PRAI and IGPS the stable and monofunctional enzymes tPRAI and tIGPS from the hyperthermophilic bacterium Thermotoga maritima (Tm) were chosen. These turned out to be easy to purify after heterologous expression in E.coli and to crystallize. These studies were also undertaken to understand the structural basis of protein thermostability. This chapter describes the production of these proteins and their purification, as well as their steady-state enzyme kinetics and stability properties.

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

Comparison of Nitrosospira strains isolated from terrestrial environments

Most of our knowledge about the physiology of ammonia-oxidizing bacteria is based on experiments with Nitrosomonas europaea, which appears to be less ubiquitous than Nitrosospira. We have isolated Nitrosospiras from widely different environments and compared their specific growth rate, substrate affinity, urease activity, temperature response, pH tolerance and cell morphology. Two of the strains had a variable morphology: the spirals were less tightly coiled than the classical Nitrosospira type and a fraction of the culture had a vibrioid appearance. These vibrioid strains were also peculiar in having a much higher apparent activation energy for ammonia monooxygenase (AMO) (129 and 151 kJ mol −1) than that of the more classical Nitrosospiras (78 and 79 kJ mol −1). The differences in morphology and activation energy were congruent with the phylogeny of the genes for 16S rRNA (Utåker et al., System. Appl. Microbiol. 18) and AMO. The response to pH in the medium was investigated for four strains. The oxidation rate at the onset of the pH exposure experiment was found to obey classical steady state enzyme kinetics, assuming that NH 3 (not NH 4 +) is the rate-limiting substrate. The calculated half saturation constants ( K s) for AMO were 6–11 μM NH 3. Growth had a narrower pH range than oxidation activity and appeared to be restricted by pH-dependent factors other than NH 3. All the isolated strains were urease positive, with a specific urease activity ranging from 60 to 158% of their specific AMO activity. The urease activity was unaffected by acetylene inhibition of the energy metabolism. The substrate affinity for one strain was found to be around 670 μM.

Comparison of Nitrosospira strains isolated from terrestrial environments.

Most of our knowledge about the physiology of ammonia-oxidizing bacteria is based on experiments with Nitrosomonas europaea, which appears to be less ubiquitous than Nitrosospira. We have isolated Nitrosospiras from widely different environments and compared their specific growth rate, substrate affinity, urease activity, temperature response, pH tolerance and cell morphology. Two of the strains had a variable morphology: the spirals were less tightly coiled than the classical Nitrosospira type and a fraction of the culture had a vibrioid appearance. These vibrioid strains were also peculiar in having a much higher apparent activation energy for ammonia monooxygenase (AMO) (129 and 151 kJ mol(-1)) than that of the more classical Nitrosospiras (78 and 79 kJ mol(-1)). The differences in morphology and activation energy were congruent with the phylogeny of the genes for 16S rRNA (Utåker et al., System. Appl. Microbiol. 18) and AMO. The response to pH in the medium was investigated for four strains. The oxidation rate at the onset of the pH exposure experiment was found to obey classical steady state enzyme kinetics, assuming that NH(3) (not NH(4)(+)) is the rate-limiting substrate. The calculated half saturation constants (K(s)) for AMO were 6-11 µM NH(3). Growth had a narrower pH range than oxidation activity and appeared to be restricted by pH-dependent factors other than NH(3). All the isolated strains were urease positive, with a specific urease activity ranging from 60 to 158% of their specific AMO activity. The urease activity was unaffected by acetylene inhibition of the energy metabolism. The substrate affinity for one strain was found to be around 670 µM.

Steady-state enzyme kinetics in the Escherichia coli periplasm: a model of a whole cell biocatalyst

This study provided analysis of in vivo enzyme kinetics in a model system which consisted of alkaline phosphatase in the periplasm of Escherichia coli. Modeling of complete substrate titration curves was achieved for a wide range of intraperiplasmic enzyme levels and outer membrane permeabilities. The results helped to identify the features most important to optimize in vivo reaction velocity. For many situations, a surprising finding was that maximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0–5 mM), the limiting step for the enzyme in the periplasm was substrate ( para-nitrophenylphosphate) diffusion through the outer membrane. In vivo reaction velocity was directly proportional to substrate concentration, outer membrane permeability, and the cell concentration. Velocity was also quite insensitive to a potent inhibitor of the enzyme. Even though diffusion-limited, periplasmic reaction velocity was quite sensitive to temperature, suggesting that the conformation of porin proteins in the E. coli outer membrane governed the average size of the pore. This model system therefore defined important features of bacterial whole cell biocatalyst design, which may also apply to other reactors using intact cells as catalysts.

Real-time measurements of dark substrate catalysis.

We have developed a novel procedure to monitor the real-time cleavage of natural unmodified peptides (dark substrates). In the competition-based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady-state enzyme kinetics for a two-substrate system, we were able to determine both Km and k(cat) values for cleavage of the dark substrate. The method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.

General Equation of Steady-State Enzyme Kinetics Using Net Rate Constants and Its Applicaiton to the Kinetic Analysis of Catalase Reaction

The steady-state velocity equation is derived for the general reaction scheme containingnkinds of enzyme species connected by a network of reversible reaction steps. The general equation is represented by the net rate constants as well as the true rate constants of the individual reaction steps. Using a general equation, equations for simpler schemes can easily be derived. Furthermore, the velocity equation expressed by net rate constants is useful for understanding the dynamic state of any reaction be it simple or complex. The generality of the presented equation is tested in a complex reaction involvingBacillus stearothermophiluscatalase I. In addition, the applicability of the general equation in analysing the reaction specificity and dynamic state of the reaction is shown.

Steady state enzyme velocities that are independent of [enzyme]: an important behavior in many membrane and particle-bound states.

The popular paradigm for biological education in kinetics involves descriptions that are appropriate for soluble enzymes. Derivations seldom present the assumptions on which the fundamental parameter of these kinetics, the site rate constant, is based. This omission can create difficulty for understanding situations where the assumptions are invalid. Membrane- and particle-bound enzymes systems provide several examples. In fact, biological organisms show macroscopic design and enzyme expression levels which suggest utilization of alternative kinetic mechanisms. The role of substrate affinity and enzyme inhibitors is greatly altered, with correlated impact on biomedical and biotechnological designs. Enzymes may perform functions such as isolation of cell contents from the environment, an action that is usually reserved for membranes. These properties can be mimicked but never perfectly replicated in purified systems. This presentation provides a description of some of these behaviors for membrane- or particle-bound enzymes, using an approach that is closely correlated with the manner in which steady state enzyme kinetics are typically presented.

Enzyme kinetics: from diastase to multi-enzyme systems

Preface Part I. Basic Steady State Enzyme Kinetics: 1. Derivation of a rate equation 2. A closer look at the basic assumptions 3. Enzyme inhibition 4. Reversible enzyme-catalyzed reactions Part II. Enzyme Reaction Sequence: 5. Multi-reactant enzymic reactions 6. Analysis of multi-reactant enzyme kinetics 7. Prediction of reaction sequence 8. Enzyme-catalyzed isotopic exchange 9. Kinetic isotope effect on steady state parameters 10. The effect of pH on enzyme kinetics Part III. Non-Hyperbolic Enzyme Kinetics: 11. The causes of non-hyperbolic enzyme kinetics 12. Analysis of non-hyperbolic enzyme kinetics 13. The effect of subunit interactions on enzyme kinetics Part IV. Control of Multi-Enzyme Systems: 14. Control of linear multi-enzyme systems 15. Control of branched multi-enzyme systems 16. Biochemical systems theory Part V. Solutions To Problems: Author index Subject index.

Expression of bovine heart fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis.

Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in KmFru6P, a 36% increase in Vmax, and a 2-fold increase in Kcat/Km of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in KmFru6P, a 2.5-fold increase in Vmax, a 12-fold increase in kcat/Km of the kinase, and a 3-fold increase in kcat/Km of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased KmFru6P and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild-type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinlsq: A Program for Fitting Kinetics Data with Numerically Integrated Rate Equations and Its Application to the Analysis of Slow, Tight-Binding Inhibition Data

Kinlsq, a Matlab-based computer program for the least-squares fitting of parameters to kinetics data described by numerically integrated rate equations, is described, and three applications to the analysis of enzyme kinetics data are given. The first application was to the analysis of a simple bimolecular enzyme plus inhibitor binding curve. The kinlsq fit to these data was essentially identical to that obtained with the corresponding analytically integrated rate equation, validating kinlsq. The second application was to the fit of a numerically integrated Michaelis-Menten model to the progress curve for dipeptidyl peptidase IV-catalyzed hydrolysis of Ala-Pro- p-nitroanilide as a demonstration of the analysis of steady-state enzyme kinetics data. The results obtained with kinlsq were compared with the results obtained by fitting this time course with the integrated Michaelis-Menten equation, and with the results obtained by fitting the (S, dP/ dt) transform of the data with the Michaelis-Menten equation. The third application was to the analysis of the inhibition of chymotrypsin by the slow, tight-binding inhibitor MeOSuc-Ala-Ala-Pro-boroPhe, data not readily amenable to other methods of analysis. These applications demonstrate how kinlsq can be used to fit rate constants, equilibrium constants, steady-state constants, and the stoichiometric relationships between components.

Electron transfer in sulfite oxidase: effects of pH and anions on transient kinetics.

Intramolecular electron transfer (ET) rates in sulfite oxidase (SO) were measured using flavin semiquinone reductants [5-deazariboflavin (dRFH.) and lumiflavin (LFH.)] generated by laser flash photolysis. Rapid bimolecular reduction of the heme by the dRF semiquinone occurred (k = 4 x 10(8) M-1 s-1 at pH 6; 1 x 10(8) M-1 s-1 at pH 9), followed by heme Fe(II) reoxidation due to intramolecular electron transfer to Mo(VI). Flash-induced difference spectra indicated that only spectral processes due to reduction and oxidation of the b-type heme prosthetic group were observed, with no detectable spectral contribution from the Mo cofactor. The extent of reoxidation decreased greatly from pH 6 to 9 (50% to 3%), as expected from the shifts in the redox potentials of the heme and Mo cofactor with pH, consistent with an electron transfer equilibrium between the two redox centers. The observed rate constant for the Fe(II) to Mo(VI) electron transfer decreased from 1650 s-1 at pH 6 to 60 s-1 at pH 9 and showed a maximum of 2400 s-1 at pH 7. Increases in salt concentration greatly decreased intramolecular ET rate constants (direct reduction by flavin semiquinone was unchanged), due to the binding of anions. Titration with the sodium salts of Cl-, SO4(2-), and H2PO4-/HPO4(2-) resulted in decreases in rate constants of intramolecular ET from 1500 s-1 to < 100 s-1 at pH 6 and 7. Similar dissociation constants were measured for the binding of these anions by flash photolysis and by steady-state enzyme kinetics using the inhibition of the sulfite/cytochrome c assay reaction for sulfite oxidase. A mechanism is proposed in which anion binding to the enzyme inhibits the rate of intramolecular electron transfer.

Stability of bilirubin oxidase in organic solvent media: A comparative study on two low‐water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a "fast" phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the "fast" phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the "slow" phase observed in water-in-oil microemulsions.

Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus.

L-Lactate dehydrogenase from Bacillus stearothermophilus (BSLDH) has been shown to change its conformation in a temperature-dependent manner in the temperature range between 25 and 70 degrees C. To provide a more detailed understanding of this reversible structural reorganization of the tetrameric form of BSLDH, we have determined in the presence of 5 mM fructose, 1,6-bisphosphate (FBP) the effect of temperature on far-UV and near-UV circular dichroism (CD), Nile red-binding to the enzyme surface, NADH binding, fluorescence polarization of fluorescamine-labeled protein, and hydrogen-deuterium exchange. In addition, we have analyzed the temperature dependence of the dimer-tetramer equilibrium of this protein by steady-state enzyme kinetics in the absence of FBP. The results obtained from these measurements at various temperatures can be summarized as follows. No changes in the secondary-structure distribution are detectable from far-UV CD measurements. On the other hand, near-UV CD data reveal that changes in the arrangements of aromatic side chains do occur. With increasing temperature, the asymmetry of the environment around aromatic residues decreases with a small change at 45 degrees C and a more pronounced change at 65 degrees C. Nile red-binding data suggest that the BSLDH surface hydrophobicity changes with temperature. It appears that decreasing the surface hydrophobicity may be a strategy to increase the protein stability of the active enzyme. We have noted significant alterations in the thermodynamic binding parameters of NADH above 45 degrees C, indicating a conformational change in the active site at 45 degrees C. The hydrodynamic volume of BSLDH is also temperature dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

4] Use of weighting functions in data fitting

This chapter discusses the importance of properly casting the fitting problem in terms of weighting functions. The chapter also discusses the consequences of improper or incorrect weighting schemes in parameter resolution and shows a specific application to the analysis of steady-state enzyme kinetics, which provides an example of general interest. The specific case of the analysis of steady-state kinetics dealt with in the chapter reveals the consequences of incorrect weighting schemes as a function of experimental error. Of particular practical interest is the fact that the Lineweaver-Burk transformation always yields the least reliable parameter estimates, regardless of the nature and extent of experimental error. A final remark should be made as to the operational aspects of weighting functions. Correct data weighting guarantees least-variance estimates of the parameter values obtained in least-squares minimization. Weighting functions can be derived from the distribution of experimental errors, and data should be analyzed in the form they are taken experimentally, using a correct weighting scheme. Data transformation leads to changes in the error distribution, and consequently demands the correct reformulation of the weighting scheme.

Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects

We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands. Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed. Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition. In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme. These enzymes exhibited no conformational change upon binding PALA. The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules. This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate. The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics. In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure.

Steady state kinetics of enzymes with substrates having multiple reaction sites

An enzyme catalysing the reaction of a substrate with multiple reaction sites may display steady state kinetics described by a Michaels-Menten equation. The Km is identical for all sites considered individually and all sites together. The maximum velocity for a single site depends on the rate constants for reaction at that site and at all of other sites.

Demonstration that spleen green hemeprotein is identical to granulocyte myeloperoxidase

The biochemical, spectroscopic, enzymatic, and redox properties of spleen myeloperoxidase, a peroxidase formerly called "spleen green hemeprotein," and granulocyte myeloperoxidase were compared by electrophoresis, amino-terminal amino acid sequences, optical and EPR spectroscopy, steady-state enzyme kinetics, and oxidation-reduction potential. The spleen enzyme exhibits properties indistinguishable from those of the granulocyte enzyme. We conclude that the spleen enzyme is indeed identical to granulocyte myeloperoxidase.