CC chemokine MIP-1 beta can function as a monomer and depends on Phe13 for receptor binding.

The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization.

Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis.

Single tryptophan residues were incorporated into each of three peptide segments that play key roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substrate binding were used to calculate binding constants and to detect the structural rearrangements that coordinate reactions at active sites for glutamine hydrolysis and PRTase catalysis. Pre-steady state kinetics of enzyme.PRPP and enzyme.PRPP.glutamine complex formation were determined from stopped-flow fluorescence measurements. The kinetics of the formation of the enzyme.PRPP complex were consistent with a model with two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzyme isomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely the rate-limiting step in the reaction of PRPP with NH(3). The pre-steady state kinetics for binding glutamine to the binary enzyme. PRPP complex could also be fit to a model involving rapid equilibrium binding of glutamine followed by an enzyme isomerization step. The changes monitored by fluorescence account for the interconversions between "end state" structures determined previously by X-ray crystallography and define an intermediate enzyme.PRPP conformer.

Purification and characterization of monomeric Escherichia coli vitamin B12 receptor with high affinity for colicin E3.

The btuB gene product from Escherichia coli is a 66.5-kDa integral outer membrane protein required for high-affinity uptake of cyanocobalamin and the translocation of E group colicins and colicin A. Efficient purification of overexpressed BtuB containing stoichiometric levels of bound lipopolysaccharide has been achieved through the extraction of the outer membrane with nonionic detergent followed by ion-exchange chromatography. Analysis of far UV circular dichroism spectra indicates a predominantly beta-sheet secondary structure (76 +/- 4%) with a low alpha-helical content (15 +/- 3%), providing the first direct evidence for secondary structure models derived from sequence and hydropathy analysis. Characterization of the octylglucoside-solubilized receptor by sedimentation equilibrium and sedimentation velocity analysis reveals a monodisperse protein-detergent complex of approximately 89 kDa with a sedimentation coefficient of 4.7 S which, after correction for bound detergent, indicates that BtuB is purified as a monomer. BtuB binds vitamin B12 with a stoichiometry of approximately 1:1, as observed by a shift in the sedimentation profile of the vitamin to the much faster velocity observed for the protein-detergent complex. The preincubation of colicin E3 with stoichiometric levels of BtuB protects susceptible strains from the lethal effects of the colicin and results in a complex with a sedimentation coefficient appropriate for a BtuB-detergent-colicin E3 complex, demonstrating that monomeric BtuB retains high affinity for this particular ligand after isolation.

The conformation of NAD+ bound to lactate dehydrogenase determined by nuclear magnetic resonance with suppression of spin diffusion.

We have reinvestigated the conformation of NAD+ bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD+ is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD+ that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both syn and anti conformations, but that binding interactions in the syn conformation are not catalytically productive.

Source of catalysis in the lactate dehydrogenase system. Ground-state interactions in the enzyme-substrate complex.

The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C(+)-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195. Thus, while the C = O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Internal chemical bonding in solutions of simple phosphates and vanadates.

The chemical bonding within structurally related phosphates and vanadates in aqueous solution is compared on the basis of vibrational frequencies obtained by classical Raman spectroscopy. To do this, an empirical relationship between the stretching frequency of P-O and P-OH or P-OR groups and bond strength is developed such that the sum of the PO bond strengths, expressed in terms of average number of electron pairs per bond, is as close as possible to 5.0 for phosphoric acid and various anions and esters thereof. The same approach is used for the corresponding vanadates. The internal bonding in phosphates involves a greater bond strength for P-OH and a smaller strength for P-O than might be expected from a simple consideration of canonical resonance forms. In vanadates, V-OH and V-O are closer to single and double bonds, respectively, than in phosphates, and the force constant for V = O is considerably smaller than for P = O, although that for V-OH and P-OH is similar. Since treating the P-O and V-O groups of simple tetrahedral phosphates and vanadates as independent diatomic oscillators provides good correlations between the respective frequencies and bond strengths, the same correlations are used to approximate the expected stretching frequencies for distorted phosphates and vanadates. The distortions considered are those that presumably characterize associative and dissociative transition states for a concerted transfer of the (PO3-) fragment of a dianionic phosphate group between donor and acceptor oxygens with similar character.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of vibrational frequencies of critical bonds in ground-state complexes and in a vanadate-based transition-state analog complex of muscle phosphoglucomutase. Mechanistic implications.

The symmetric stretching frequency of the P-O bonds of the enzymic phosphate group in muscle phosphoglucomutase was measured via 16O/18O Raman difference spectroscopy. This frequency, and its shift on isotopic substitution, is characteristic of a dianionic phosphate ester. The P-O stretching frequency is not detectably altered by the binding of the metal ion activators Mg2+, Zn2+, or Cd2+ nor by the subsequent binding of glucose phosphate. Hence, a binding-induced distortion/polarization of the enzymic phosphate group in the ground state, or enzyme-substrate complex, cannot serve as a rationale for the large value of kcat in the phosphoglucomutase reaction. By contrast, the stretching frequency of the V-O bonds within a vanadate group bound at the same site in the transition-state analog complex involving glucose 1-phosphate 6-vanadate is much lower than for a normal dianionic vanadate. This low V-O stretching frequency is best rationalized in terms of the extensive polarization of all three nonbridging oxygens of the vanadate ester dianion plus the formation of a weak, fifth bond to the vanadium atom. This distortion/polarization of the VO3(2-) group depends on the metal ion activator, since it is largely abolished, and the involvement of the fifth ligand eliminated, by substitution of Li+ for Mg2+ at the metal activation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of nonmetabolizable opines by Agrobacterium tumefaciens.

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.

Characterization of vanadate-based transition-state-analogue complexes of phosphoglucomutase by spectral and NMR techniques.

Near ultraviolet spectral studies were conducted on two inhibitor complexes obtained by treating the dephospho form of the phosphoglucomutase.Mg2+ complex with inorganic vanadate in the presence of either glucose 1-phosphate [cf. Percival, M. D., Doherty, K., & Gresser, M. J. (1990) Biochemistry (first of four papers in this issue)] or glucose 6-phosphate. Part of the spectral differences between the two inhibitor complexes arises because the glucose phosphate moiety in the complex derived from glucose 1-phosphate binds to the enzyme in a different way from the glucose phosphate moiety in the complex derived from glucose 6-phosphate and because these alternative binding modes produce different environmental effects on the aromatic chromophores of the dephospho enzyme. These spectral differences are strikingly similar to those induced by the binding of glucose 1-phosphate and glucose 6-phosphate to the phospho enzyme--which shows that the glucose 1-phosphate and glucose 6-phosphate moieties occupy positions in the inhibitor complexes closely related to those that they occupy in their respective catalytically competent complexes. This binding congruity indicates that in the inhibitor complexes the oxyvanadium grouping is bound at the site where (PO3-) transfer normally occurs. 31P NMR studies of the phosphate group in these complexes also provide support for this binding pattern. A number of other systems based on compounds with altered structures, such as the deoxysugar phosphates, or systems with different compositions, as in the case of the metal-free enzyme or of the glucose phosphates plus nitrate, also were examined for evidence that complexes analogous to the inhibitor complexes were formed, but none was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Remote nitrogen-15 isotope effects on addition of cyanide to NAD.

The reversible reaction NAD + CN(-)----NAD-CN was examined for remote secondary 15N isotope effects caused by isotopic substitution at the ring nitrogen of the nicotinamide group. These were compared with analogous effects for dehydrogenase-catalyzed reactions, since both cyanide and the hydride ion add at the N-4 position of the nicotinamide ring. The 15N effects on the rate constants for the forward and reverse processes were examined directly by conducting both the normal and isotopic reactions simultaneously under carefully controlled conditions in the sample and reference cells of a dual-beam spectrophotometer. In both cases, the 15N kinetic isotope effect differed from 1.00 by considerably less than 0.01. The 15N equilibrium isotope effect, 15K, was obtained as the ratio of equilibrium constants measured separately with natural-abundance and labeled NAD by using a concentration jump procedure [1.004 +/- 0.002 (cyanide addition)]. A similar value for 15K of 1.010 +/- 0.008 was obtained in an analogous manner for the reaction catalyzed by lactate dehydrogenase: NAD + lactate----pyruvate + NADH + H+. The latter value is significantly smaller than a previously reported value obtained from kinetic studies [1.044 +/- 0.012; Cook, P. F., Oppenheimer, N. J., & Cleland, W. W. (1981) Biochemistry 20, 1817]. The present value also is smaller than might be expected for a change in bond order from 4 to 3 [Cleland, W. W. (1980) Methods Enzymol. 64, 104-125] on the basis of the canonical resonance structures for NAD and NADH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acceleration of the NAD cyanide adduct reaction by lactate dehydrogenase: the equilibrium binding effect as a measure of the activation of bound NAD.

The binary complex of NAD and lactate dehydrogenase reacts reversibly with cyanide to produce a complex (E X NAD-CN) whose noncovalent interactions are similar to those in the E X NADH complex (where E is one-fourth of the tetrameric dehydrogenase). The reaction apparently is a simple bimolecular nucleophilic addition at the 4 position of the bound nicotinamide ring; viz., cyanide does not bind to the enzyme prior to reaction. The value of the dissociation constant for E X NAD-CN is about 1 X 10(-6) M and is independent of pH over the range of 6-8. The equilibrium constant for the reaction of cyanide with E X NAD is about 400-fold larger than that for the nonenzymic process after a statistical correction. This increment in Ke is accounted for by a 220-fold increase in the rate of the forward enzymic reaction (20 M-1 s-1) as compared with an approximately 2-fold decrease for the reverse process (9 X 10(-5) s-1). Thus, the increased value of the rate constant for bond formation in the enzymic reaction is attributed to an equilibrium binding effect that is translated almost entirely into a rate effect on that step (bond formation). Since the nonenzymic reaction is sensitive to solvent composition, this equilibrium binding effect likely is produced by environmental effects at the nicotinamide/dehydronicotinamide part of the coenzyme binding site on the enzyme.

The lactate dehydrogenase catalyzed pyruvate adduct reaction: simultaneous general acid-base catalysis involving an enzyme and an external catalyst.

The pH dependence of the reaction catalyzed by lactate dehydrogenase, where pyruvate adds covalently to NAD to yield a NAD-Pyr adduct, together with published data on the pH dependence of parameters in the normal redox reaction suggests similar binding modes for enolpyruvate and lactate in their complexes with E X NAD (where E is one-fourth of the tetramer), for ketopyruvate in its complexes with the protonated species, E X H X NAD and E X H X NADH, and for the NAD--Pyr adduct and NADH plus pyruvate in their complexes with E X H. These similarities, together with previous data, suggest a reaction scheme for the formation of the enzyme-adduct complex that includes the relevant proton-transfer steps. Seven different amine chloride buffers were used in a study of the reverse adduct reaction, i.e., the decomposition of E X H X NAD--Pyr. These act with varying efficiencies as external general acid catalysts; the enzyme apparently acts as a (internal) general base. The involvement of the amine chloride buffers as external general catalysts is supported by the concentration dependence of the buffer effect, by a Brönsted plot, and by solvent deuterium isotope effects. The involvement of the enzyme as an internal general catalyst is inferred from the pH dependence of the reaction and the identities of the nearby groups in the E X H X NAD--Pyr complex (from crystallographic studies). The dependence of the adduct reaction on chloride concentration indicates the presence of dead-end inhibitor complexes of E X H X Cl and E X H X NAD X Cl. Chloride also accelerates the decomposition of the adduct in the complex E X H X NAD--Pyr by binding to this complex.

On the origin of the lactate dehydrogenase induced rate effect.

To evaluate the ability of lactate dehydrogenase to facilitate the bond making/breaking steps for both the addition of pyruvate enol to NAD (pyruvate adduct reaction) and the normal redox reaction, the ability of the enzyme to facilitate the tautomerization of bound pyruvate is assessed. In addition, the equilibrium constants for the adduct reaction are obtained for both bound and free reactants from the ratio of the rate constants in the forward and reverse reactions (at pH 7). The latter comparison indicates that the enzyme facilitates bond making/breaking in the (forward) pyruvate adduct reaction by a factor of about 10(11) M. Similar comparisons suggest that reactant immobilization accounts for about 1000 M of this 10(11) M rate effect. Since the (pH-independent) rate constant for the ketonization of bound pyruvate enol assisted by the external buffer, imidazolium ion, is 2 X 10(7) M-1 s-1 and the corresponding rate constant for free pyruvate enol, again assisted by imidazolium ion, is 35 M-1 s-1 [Burger, J. W., II, & Ray, W. J., Jr. (1978) Biochemistry 17, 1664], the enzyme facilitates the bond making/breaking steps associated with the conversion of bound HO-C less than to bound O = C less than by a factor of about 10(6)-fold. The product of the above two rate enhancement factors and the rate factor suggested previously for the environmental effect on NAD produced by its binding to lactate dehydrogenase, 100-fold, is 10(11) M, and it accounts for the bond making/breaking effects exerted by the enzyme in the pyruvate adduct reaction. The rate constant for oxidation of ethanol (a model for lactate) by 1-methylnicotinamide (a model for NAD) is about 5 X 10(-12) M-1 s-1 at 25 degrees C in pure ethanol (delta H for this reaction is about 30 kcal/mol). The ratio of the rate constants for E X NAD X Lac----E X NADH X Pyr and the above model reaction is estimated as about 10(14) M in water; i.e., the LDH-induced rate effect is about 10(14) M. The product of the values for the above rate factors for the normal redox reaction is about 10(12) M. Although the value of this product is less certain than that for the adduct reaction, these rate factors do account for much of the LDH-induced rate effect.

Mechanistic study of the addition of pyruvate to NAD+ catalyzed by lactate dehydrogenase.

The binary complex of NAD+ and dogfish A4 lactate dehydrogenase reacts reversibly with pyruvate enol to produce an inactive, enzyme-adduct complex, in which the nicotinamide and pyruvate moieties are linked by means of a covalent bond. This process is examined in both the forward and reverse directions as a function of reactant and buffer concentrations at pH 7, under conditions where the enolization of pyruvate is at equilibrium, and the involvement of complexes with stoichiometry E.NAD, E.NAD.PyrE, and E.NAD.PyrK is defined. (The subscripts, E and K, indicate the enol and keto forms of pyruvate.) One pathway for formation of the adduct complex involves the prior formation of the E.NAD.PyrE complex from E.NAD and pyruvate enol; the alternative pathway involves formation of the same complex via the enolization of E.NAD.PyrK, a process that is catalyzed by an external (nonenzymic) base. The possible use of the adduct reaction as a model for the normal enzymic reaction is considered.