Determination of dissociation constants of crystalline-alpha-chymotrypsin complexes

As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl-l-phenylalanine, and N-formyl-l-p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl-l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl-p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme.The information obtained from these simple equilibrium measurements is in agreement with X-ray diffraction studies. The approach is, therefore, capable of determining whether or not compounds bind to the active site of a crystalline enzyme, and whether the occupancy of this site is sufficient for structure determinations using X-ray diffraction methods.     

Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD(+) in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pK(a) approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185-4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD(+) release from the enzyme-NAD(+)-ethanol product complex. This finding permits calculation of K(3), the dissociation constant for ethanol from the enzyme-NAD(+)-ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613-627). The calculations show that K(3) varies very little with pH in the range 5.95-8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme-NADH-acetaldehyde]/ [enzyme-NAD(+)-ethanol] at equilibrium. The values obtained were in the range 0.11+/-0.04, and this value together with estimates of K(3) was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.     

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.     

Acid dissociation constants of glycopeptides.

Glycopeptides containing mainly four amino acid residues in the sequence Asn-Leu-Thr-Ser, with small amounts of additional amino acid residues, were isolated from enzymic hydrolysates of hen's-egg albumin. Heterogeneity of the carbohydrate moiety was confirmed. Acid-base titrations showed that the alpha-amino group has a pKa value of 6.43 at 25 degrees C. The standard free engery and entropy changes associated with the ionization at 25 degrees C were 37.2kJ-mol-1 and -0.014kJ-mol-1- K-1 respectively. The complications arising in the interpretation of titration curves of the glycopeptides, which are heterogeneous with respect to the peptide chain, were considered and discussed in the light of the earlier suggestion that the titration curve of the glycopeptide might be interpreted as being due in part to a structure in which the hydroxyl group of the threonine residue is hydrogen-bonded to the beta-aspartamido oxygen atom [Neuberger & Marshall (1968) in Symposium on Foods - Carbohydrates and their Roles (Schultz, H.W., Cain, R.F. & Wrolstad, R.W., eds.), pp. 115-132, Avi Publishing Co., Westport, CT]. It is concluded that either the glycopeptides do not contain a hydrogen bond of that type, or, if they do, that it cannot be recognized by acid-base-titration studies.     

Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Determining ligand dissociation constants of binary and ternary complexes from the kinetics of enzyme inactivation

Adenosine 5-phosphosulfate (APS) kinase from Penicillium chrysogenum is irreversibly inactivated by trinitrobenzene sulfonate in a pseudo-first order process. Under standard assay conditions kapp was 1.9 X 10(-3) s-1. Saturating MgATP or MgADP decreased Kapp to a limit of 4.1 X 10(-4) s-1. There are several explanations for the partial protection, including the presence of two essential lysyl side chains, only one of which is at the active site. Analysis of the inactivation kinetics by means of linear plots derived for partial protection yielded dissociation constants for E X MgATP (Kia) and E X MgADP (Kiq) of 2.9 mM and 1.8 mM, respectively. Low concentrations of APS alone provided no protection against trinitrobenzene sulfonate inactivation, but in the presence of 1 mM MgADP, as little as 2 microM APS provided additional protection while 100 microM APS reduced kapp to the limit of 4.1 X 10(-4) s-1. The results confirm the formation of a dead end E X MgADP X APS proposed earlier as the cause of the potent substrate inhibition by APS. Linear plots of 1/delta k versus 1/[MgADP] at different fixed [APS] and of 1/delta k versus 1/[APS] at different fixed [MgADP] were characteristic of the ordered binding of MgADP before APS (or the highly synergistic random binding of the two ligands). The true APS dissociation constant of the dead end E X MgADP X APS complex (K'ib) was determined to be 1.9 microM. From the value of K'ib and the previously reported value of KIB (apparent inhibition constant of APS as a substrate inhibitor of the catalytic reaction at saturating MgATP), the ratio of the MgADP and PAPS release rate constants (k4/k3) was calculated to be 11. Inactivation kinetics was used to study the effects of Mg2+ and high salt on ADP and APS binding. The results indicated that free ADP binds to the enzyme more tightly than does MgADP at low ionic strength. High salt decreased free ADP binding, but had little effect on MgADP binding. APS binds more tightly to E X MgADP in the absence or presence of salt than to E X ADP.     

Relations between the dissociation constants of dibasic acids.

The relationships between the molecular pK values of a dibasic acid, its titration pK values and the pH values at which the concentration of monohydronated species is half-maximal are presented. These enable any pair of the values to be found from any other pair.     

Crystallization and preliminary analysis of enzyme-substrate complexes of pyruvate kinase from rabbit muscle.

Pyruvate kinase from rabbit muscle has been crystallized in a form suitable for high resolution X-ray analysis. Complexes of the enzyme with Mn2+ and either pyruvate or oxalate crystallize from solutions of polyethyl-eneglycol 8000 at pH 6.0. Crystals obtained from solutions of the complexes with pyruvate or oxalate appear isomorphous and belong to the triclinic space group P1. The crystals have unit cell dimensions a = 83.3(4) A, b = 109.4(6) A, c = 145.7 (7) A, alpha = 94.9 degrees, beta = 93.6 degrees, gamma = 112.2 degrees. These crystals diffract to better than 2.4 A resolution and are stable in the X-ray beam for at least 20 hr. Electron paramagnetic resonance measurements on a single crystal show that Mn2+ is bound to the crystalline protein.     

Rat kidney porphobilinogen deaminase kinetics. Detection of enzyme-substrate complexes

BACKGROUND AND AIMS: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions. METHODS: Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0-20 min) and over a wide range of substrate concentrations (0.8-66 microM). RESULTS: Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS-PAGE, Western blot and isoelectric focusing (pI 4.9). Its molecular mass is 40 +/- 2.3 kDa, determined by SDS-PAGE and 39.8 +/- 2 kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis-Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K(1) = 2.08 +/- 0.01 microM and K(2) = 0.102 +/- 0.003 microM. CONCLUSION: The values of these constants fulfil the restriction 4K(2) < or = K(1)(2), necessary for the occurrence of isoenzymes, interpreted in this work as enzyme-substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme-substrate intermediates formed during PBG-D reaction.     

Determination of rate constants for nucleotide dissociation from Na,K-ATPase.

A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.     

Computational method for relative binding energies of enzyme-substrate complexes.

A computational method for estimating the relative binding free energies of enzyme-substrate complexes is described that combines electrostatic and solvation models and X-ray crystallographic data. The polar contribution is evaluated by the Poisson-Boltzman equation. The nonpolar contribution is evaluated by solvent transfer data and surface area calculations. This algorithm was used to calculate the relative binding energies of 63 pairs of nine different mutant proteins with seven different substituted R-malate substrates of Escherichia coli isocitrate dehydrogenase. Comparison of calculated values with the experimentally observed values shows a high degree of correlation.     

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.     

A spectrophotometric resin competition procedure to determine dissociation constants for metal ion-nucleotide complexes

The resin competition method for determining the dissociation constants of metal ion-nucleotide complexes was modified to take into account the fact that some metal nucleotide complexes are anionic and thus, like the free nucleotide, will bind to the resin. A simple, rapid spectrophotometric titration procedure is given for the determination of both the metal ion-nucleotide complex dissociation constant and the ratio of the affinities of the nucleotide and its complex for the resin.