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L D Kwiatkowski

Publications and source records attributed to L D Kwiatkowski.

17 recordsLinked to original sources
PubMed2001

Mutational effects at the subunit interfaces of human hemoglobin: evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the alpha 1 beta 2 interface.

A set of variant human hemoglobins, each with an Ala or Gly substitution at a single residue, has been prepared, and the kinetics of their reactions with carbon monoxide have been measured. This reaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. The magnitudes of the effects of the mutations on CO combination vary widely, and, with the exception of beta Y145, the residues with the most significant effects on these kinetics are found in the hinge region of the alpha 1 beta 2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heme on both alpha or both beta subunits, were prepared for beta W37E, beta W37A, alpha Y140G, and alpha Y140A, hinge region variants causing large kinetic changes, and for beta Y145G. Such hybrids permit measurements of the kinetics of CO binding to only the heme-containing alpha or beta subunits within the unliganded hemoglobin tetramer. Mutations at beta 37 and alpha 140 have global effects on the T state, increasing the rates of CO binding to both types of subunits. Mutation of beta Y145 has a large effect on the beta subunits in the deoxygenated T state, but very little effect on the alpha subunits. Oxygen equilibria measurements on the crystalline T state of beta W37E also indicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of the variant hemoglobins in solution are sensitive to numerous variables besides the properties of the deoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause the largest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the alpha 1 beta 2 interface.

Amino Acid Substitution↗
PubMed2001

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces.

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.

Amino Acid Substitution↗
PubMed1998

Preparation and kinetic characterization of a series of betaW37 variants of human hemoglobin A: evidence for high-affinity T quaternary structures.

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.

Amino Acid Substitution↗
PubMed1998

Thermodynamic studies on the equilibrium properties of a series of recombinant betaW37 hemoglobin mutants.

In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].

Amino Acid Substitution↗
PubMed1997

Structure and oxygen affinity of crystalline des-his-146beta human hemoglobin in the T state.

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146beta human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146beta, the COOH-terminal histidine residues of the beta chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146beta results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141alpha hemoglobin showed that removal of the COOH termini of the alpha chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146beta in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141alpha than those associated with His-146beta.

Computer Simulation↗
PubMed1995

Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state.

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extremely complex problem due to potential differences of protein conformational flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the structure and the function of a protein in the same crystalline environment. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the alpha-chain COOH-terminal residue, Arg141 alpha) have been studied in the same crystal form by high resolution X-ray diffraction methods and by polarized absorption microspectrophotometry. Specifically, the crystal structure of deoxygenated desArg human hemoglobin has been refined at a 2.1 A resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141 alpha and all of the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains which include an increase in the conformational flexibility of both the NH2 and COOH-terminal peptides. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterization by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fold greater than that of crystals of human hemoglobin A. There is no alkaline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes of the crystals differ by 25%, indicating heterogeneity in the affinities of the oxygen binding sites. This finding and the measured Hill coefficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity and the implied cooperativity are unknown.

Arginine↗
PubMed1994

Isolation and stability of partially oxidized intermediates of carp hemoglobin: kinetics of CO binding to the mono- and triferric species.

The monoliganded and triliganded forms of the asymmetric valency hybrids of carp hemoglobin were isolated using high-performance liquid chromatography. These partially oxidized hybrids were shown to be sufficiently stable to permit the measurement of the kinetics of CO binding. The effects of protons and inositol hexaphosphate on the rates of these reactions were examined. The kinetics of CO recombination with these partially oxidized derivatives were compared to the kinetics of CO binding to the fully ferrous molecule. To a first approximation, the kinetic behavior of the monoferric derivative was consistent with a small shift in the T<==>R equilibrium in favor of the R state. The presence of three ferric ligands resulted in a still greater shift in the conformational equilibrium in favor of the R state. The kinetic behavior of the triferric molecule was similar, but not identical, to that of a fully ferrous molecule which is triliganded with CO. The properties of both asymmetric valency hybrids were responsive to the nature of the ligand; i.e., the rate of CO binding was increased more by the presence of cyanide on the ferric hemes than by water. Not all of the data could be accommodated within the two-state model. For example, there was evidence of an altered T state in the case of the tricyanomet derivative at low pH in the presence of inositol hexaphosphate.

Animals↗
PubMed1993

The effect of 75% glycerol on the oxygen binding properties of carp hemoglobin.

At pH 6 in the presence of inositol hexaphosphate, IHP, conditions where ligand-saturated carp hemoglobin is already in the low affinity T state, the addition of glycerol has little effect on affinity and ligand binding remains noncooperative. At all other pH values examined, with and without IHP, the effect of glycerol is to lower oxygen affinity possibly by shifting the equilibrium between the T state and the high affinity R state in the direction of the T state. Although glycerol does not appear to have an appreciable effect on the T state itself, a small effect on the R state cannot be excluded by our data.

Allosteric Regulation↗
PubMed1993

Effect of chloride on oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure.

Oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure has been investigated by polarized absorption microspectrophotometry. These crystals were grown from poly(ethylene glycol) solutions containing low concentrations of salt. In the absence of chloride, they have a significantly higher oxygen affinity than crystals of human hemoglobin A grown in a similar manner, and exhibit Hill coefficients lower than 1. There is no Bohr effect from pH 6 to 9. We have found that chloride decreases the oxygen affinity of Hb Rothschild crystals, an effect which is absent in crystals of HbA. This dependence of affinity on chloride is almost certainly associated with the chloride binding sites which have been localized crystallographically at the mutant arginine residues (Kavanaugh et al., 1992). Since chloride binding appears to lower the oxygen affinities of both the alpha and beta chains, the linkage between the binding of oxygen and the dissociation of chloride results in significant cooperativity in oxygen binding to the crystals.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin↗
PubMed1987

Contribution of arginine (HC3) 141 alpha to the Bohr effect of the fourth binding step in the reaction of ligand with human hemoglobin.

A few years ago we reported that histidine (HC3) 146 beta plays a major role in the pH-dependent properties of the R-state of human hemoglobin, accounting for close to 50% of the R-state Bohr effect. We have extended these studies by examining the role of arginine 141 alpha, another group known to affect the overall Bohr effect. We have compared the pH dependencies of the rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l4 and l'4, respectively, for native hemoglobin A (HbA) and a control reconstituted HbA, and des-(Arg 141 alpha) HbA, the hemoglobin molecule resulting from the enzymatic removal of the C-terminal arginine of the alpha-chain of human Hb. From these kinetic constants the pH dependence of L4, the affinity constant for the fourth carbon monoxide molecule, has been estimated. We find that the removal of arginine 141 alpha reduces the pH dependence of log L4 by about 80% between pH 6 and 8, where the alkaline Bohr effect normally occurs. The sum of the effects of the removal of His 146 beta and of Arg 141 alpha is greater than 100%. This suggests that at least one of these modifications alters the contributions of other residues of this Bohr effect.

Arginine↗
PubMed1986

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder.

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p50(CO) of 100 mmHg at 15 degrees C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10(3) M-1 X s-1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.

Air Sacs↗
PubMed1982

The contribution of histidine (HC3) (146 beta) to the R state Bohr effect of human hemoglobin.

The role of histidine (HC3) 146 beta in the previously established pH-dependent properties of the R state of human hemoglobin has been investigated. The rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l4 and l'4, have been determined as a function of pH for hemoglobin A and des-(His 146 beta) hemoglobin A. From these kinetic parameters, the value of L4, the affinity constant for the 4th carbon monoxide molecules, has been calculated according to the equation L4 = l'4/l4. In addition, the effect of removal of histidine 146 beta or its replacement by arginine (hemoglobin Cochin-Port Royal) on k4, the rate of oxygen dissociation from fully liganded hemoglobin, has been determined as a function of pH. Removal of histidine 146 beta reduces the pH dependence of L4 by 47%. At the same time, it produces a similar, 45%, reduction in the pH dependence of kr. Replacement of histidine 146 beta by arginine reduces the pH dependence of k4 by 23% and that of l'4 by about 30%. These chemical modifications cause reductions in the R state Bohr effect which are remarkably similar in magnitude to the reduction which they produce in the overall Bohr effect. These results indicate that histidine 146 beta controls a major fraction of the R state Bohr effect as well as being a major participant in the overall and T state Bohr effects.

Carbon Dioxide↗
PubMed1981

Kinetic mechanism of the Cu(II) enzyme galactose oxidase.

The steady-state kinetics of four redox reactions catalyzed by galactose oxidase have been determined. The alcohol substrate used in each case was galactose; the four oxidant substrates used were O2, IrCl6(2)-, porphyrexide, and Fe(CN)6(3)-. With the exception of the last reagent, saturation behavior is exhibited by all substrates. Double reciprocal plots of rate data obtained varying one substrate at various concentrations of the other are intersecting for all parsi that exhibited saturation behavior. Thus, these reactions are kinetically sequential processes involving single central complexes. These complexes involve enzyme, galactose, and one molecule of oxidant, whether or not the oxidant is a one- or two-electron acceptor. This result indicates that for one-electron oxidants, an enzyme.alcohol-derived radical species may exist as a transient prior to the reaction of the second electron equivalent of oxidant. A similar substrate radical.O2- transient is postulated in the reaction involving O2. The inhibition by H2O2 has also been studied in detail. H2O2 apparently binds to the enzyme at two sites. The nature of alcohol and O2 binding to the enzyme Cu(II) is discussed in light of these kinetic results.

Binding Sites↗