Inhibition of beta-lactamases by monocyclic acyl phosph(on)ates

The cyclic acyl phosph(on)ates, 1-hydroxy-5-phenyl-2,6-dioxaphosphorinone(3)-1-oxide, its 4-phenyl isomer, and the phosphonate (2-oxo) analogue of the latter inhibited typical class A (TEM-2) and class C (Enterobacter cloacae P99) beta-lactamases in a time-dependent fashion. No enzyme-catalyzed turnover was detected in any case. The interactions occurring were interpreted in terms of the reaction scheme E + I left arrow over right arrow EI left arrow over right arrow EI', where EI is a reversibly formed noncovalent complex, and EI' is a covalent complex. Reactions of the cyclic phosphates with the P99 beta-lactamase were effectively irreversible, while that of the 4-phenyl cyclic phosphate with the TEM beta-lactamase was slowly reversible. The 4-phenyl cyclic phosphate was generally the most effective inhibitor, both kinetically and thermodynamically, with second-order rate constants of inactivation of both enzymes around 10(4) s(-1) M(-1). This compound also bound noncovalently to both enzymes, with dissociation constants of 25 microM from the P99 enzyme and 100 microM from the TEM. It is unusual to find an inhibitor equally effective against the TEM and P99 enzymes; the beta-lactamase inhibitors currently employed in medical practice (e.g., clavulanic acid) are significantly more effective against class A enzymes. The results of lysinoalanine analysis after hydroxide treatment of the inhibited enzymes and of a (31)P nuclear magnetic resonance spectrum of one such complex were interpreted as favoring a mechanism of inactivation by enzyme acylation rather than phosphylation. Molecular modeling of the enzyme complexes of the 4-phenyl phosphate revealed bound conformations where recyclization and thus reactivation of the enzyme would be difficult. The compounds studied were turned over slowly or not at all by acetylcholinesterase and phosphodiesterase I.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

Biochemical switching device: biomimetic approach and application to neural network study.

There are many examples of enzymes that share substrates or cofactors in a cyclic manner. Techniques have been developed that use cyclic enzyme systems to assay quantitatively small amounts of biochemical substances (cofactor, substrate), however, only a few studies of the control of these systems have been published. The author previously showed with computer simulations that cyclic enzyme systems have the reliability of ON-OFF types of operation (McCulloch-Pitts' neuronic equation) capable of storing short-memory, and the applicability for a switching circuit in a biocomputer. This paper introduces a unique switching mechanism of cyclic enzyme system (basic switching element), and next, building the integrated biochemical switching system being composed of the basic switching element, shows the physiological phenomenon termed 'selective elimination of synapses' generally produced as a result of low-frequency train of electrical stimuli to the synapses (Kuroda, Y. 1989) Neurochem. Int. 14, 309-319).     

The energy transduction mechanism of Na,K-ATPase studied with iron-catalyzed oxidative cleavage.

This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects of E1 left arrow over right arrow E2 conformational transitions (Goldshleger, R. , and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E2 and E1 conformations are characterized by formation or relaxation of interactions within the alpha subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E1 left arrow over right arrow E2 and E1P left arrow over right arrow E2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.     

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.     

Enzymatic synthesis of blood group A and B trisaccharide analogues

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.     

Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.     

Comparative specificity of porcine pancreatic kallikrein and bovine pancreatic trypsin. Importance of interactions N-terminal to the scissible bond

Hydrolyses catalyzed by bovine pancreatic trypsin and porcine pancreatic kallikrein were studied using synthetic peptide substrates of the type E chi-L chi 2-L chi 1 decreases Y and E chi-L chi 3-L chi 2-L chi 1 decreases Y with L chi 1 = Arg defining the hydrolysis position (indicated by the arrow). The leaving moiety Y was -OCH3, -NH-C6H4-p-NO2 and -Ala-NH2. Insight into interactions occurring between the active site of the enzymes and the acyl moiety of the substrates was gained by studying the influence on hydrolysis rate of structural variation of residues L chi 2 and L chi 3. Parallel analyses of the hydrolyses of the ester, anilide, and peptide substrates having the same acyl moiety considerably facilitated the interpretation of the kinetic data. Trypsin, but not kallikrein, displayed high reactivity even with relatively short substrates. Ac-Ala-Arg-Ala-NH2, for example, was a better substrate for trypsin than for kallikrein by a factor of 1.3 X 10(4) in terms of kcat and 5.9 X 10(4) in terms of kcat/Km. Reactivity differences of such magnitude were related to two main differences in enzyme-substrate interactions: the interaction of the arginine side chain of the substrate with the specificity pocket of the enzyme is optimal for trypsin but poor for kallikrein and the number of hydrogen bonds formed by the enzyme with the backbone section of the substrate on both sides of the specific residue is larger in the case of trypsin. The latter difference is found to be related to the structure of amino-acid residue 192 which is glutamine in trypsin and methionine in kallikrein.     

Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase alpha subunit

The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, K-ATPase alpha subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+ and K+ affinities (K1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na+ acting at the extracellular space (Argüello et al. J. Biol. Chem. 271, 24610-24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzymes, while their activation by K+ did not appear affected. Although the Na+ K1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, nHill = 0.75; Tyr771Ser, nHill = 0.92; Control, nHill = 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+ dependence (nHill = 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 left and right arrow E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na+ binding and occlusion without participating in K+-enzyme interactions.     

Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A (1)H-NMR and electronic absorbance study

The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N long left and right arrow X long left and right arrow D, where X represents a disordered globin structure (occurring at approximately 4 M urea) that still binds the heme.     

Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution

The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.     

Cyanine dyes as intercalating agents: kinetic and thermodynamic studies on the DNA/Cyan40 and DNA/CCyan2 systems

The interaction of cyanines with nucleic acids is accompanied by intense changes of their optical properties. Consequently these molecules find numerous applications in biology and medicine. Since no detailed information on the binding mechanism of DNA/cyanine systems is available, a T-jump investigation of the kinetics and equilibria of binding of the cyanines Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with CT-DNA is performed at 25 degrees C, pH 7 and various ionic strengths. Bathochromic shifts of the dye absorption band upon DNA addition, polymer melting point displacement (DeltaT = 8-10 degrees C), site size determination (n = 2), and stepwise kinetics concur in suggesting that the investigated cyanines bind to CT-DNA primary by intercalation. Measurements with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) reveal fair selectivity of CCyan2 toward G-C basepairs. T-jump experiments show two kinetic effects for both systems. The binding process is discussed in terms of the sequence D + S left arrow over right arrow D,S left arrow over right arrow DS(I) left arrow over right arrow DS(II), which leads first to fast formation of an external complex D,S and then to a partially intercalated complex DS(I) which, in turn, converts to DS(II), a more stable intercalate. Absorption spectra reveal that both dyes tend to self-aggregate; the kinetics of CCyan2 self-aggregation is studied by T-jump relaxation and the results are interpreted in terms of dimer formation.     

Interleukin-1 beta folding between pH 5 and 7: experimental evidence for three-state folding behavior and robust transition state positions late in folding

The thermodynamic stability and folding kinetics of the all beta-sheet protein interleukin-1beta were measured between 0 and 4 M GdmCl concentrations and pH 5-7. Native interleukin-1beta undergoes a 3.5 kcal/mol decrease in thermodynamic stability, Delta, as pH is increased from 5 to 7. The native state parameter m(NU), measuring protein destabilization/[GdmCl], remains constant between pH 5 and 7, indicating that the solvent-exposed surface area difference between the native state and unfolded ensemble is unchanged across this pH range. Similarly, pH changes between 5 and 7 decrease only the thermodynamic stability, DeltaG(H)2(O), and not the m-values, of the kinetic intermediate and transition states. This finding is shown to be consistent with transition state configurations which continue to be the high-energy configurations of the transition state in the face of changing stability conditions. A three-state folding mechanism U right arrow over left arrow I right arrow over left arrow N is shown to be sufficient in characterizing IL-1beta folding under all conditions studied. The m-values of refolding transitions are much larger than the m-values of unfolding transitions, indicating that that the fast, T(2) (U right arrow over left arrow I), and slow, T(1) (I right arrow over left arrow N), transition states are highly similar to the intermediate I and native state N, respectively. Many of the folding properties of interleukin-1beta are shared among other members of the beta-trefoil protein family, although clear differences can exist.     

Calcium dependence of Pi phosphorylation of sarcoplasmic reticulum Ca2+-ATPase at low water content: water dependence of the E2-->E1 conversion.

Enzymes entrapped in reverse micelles can be studied in low-water environments that have the potential of restricting conformational mobility in specific steps of the reaction cycle. Sarcoplasmic reticulum Ca2+-ATPase was incorporated into a reverse-micelle system (TPT) composed of toluene, phospholipids, Triton X-100 and varying amounts of water (0.5-7%, v/v). Phosphorylation of the Ca2+-ATPase by ATP required the presence of both water and Ca2+ in the micelles. No phosphoenzyme (EP) was detected in the presence of EGTA. Phosphorylation by Pi (inorganic phosphate) in the absence of Ca2+ was observed at water content below that necessary for phosphorylation by ATP. In contrast to what is observed in a totally aqueous medium, EP formed by Pi was partially resistant to dephosphorylation by Ca2+. However, the addition of non-radioactive Pi to the EP already formed caused a rapid decrease in radiolabelled enzymes, as expected for the isotopic dilution, indicating the existence of an equilibrium (E+Pi<-->EP). Phosphorylation by Pi also occurred in TPT containing millimolar Ca2+ concentrations in a range of water concentrations (2-5% v/v). The substrates p-nitrophenyl phosphate, acetyl phosphate, ATP and GTP increased the EP level under these conditions. These results suggest that: (1) the rate of conversion of the ATPase conformer E2 into E1 is greatly reduced at low water content, so that E2-->E1 becomes the rate-limiting step of the catalytic cycle; and (2) in media of low water content, Pi can phosphorylate both E1Ca and E2. Thus, the effect of enzyme hydration is complex and involves changes in the phosphorylation reaction at the catalytic site, in the equilibrium between E2 and E1 conformers, and in their specificity for substrates.     

Doxorubicin inhibits the phosphate-transport protein reconstituted in liposomes. A study on the mechanism of the inhibition

Using Thr(P)-inhibitor-1 and Ser(P)-casein as substrates, studies on the activation of calcineurin purified from bovine brain have been carried out. The phosphatase requires the synergistic action of Ca2+, calmodulin and another divalent cation (Mg2+, Mn2+, Co2+ or Ni2+, but not Zn2+) for full expression of its activity. Ca2+ and Ca2+ X calmodulin act as allosteric activators to transform the phosphatase to a relaxed conformation, while Mg2+ acts solely as a cofactor for the catalytic action of the enzyme. In addition to their function as cofactors for catalysis, transition metal ions can also substitute for Ca2+ as allosteric activators. Ca2+ and calmodulin exert their activating effects mainly by increasing the Vm of the phosphatase reaction with little effect on the Km values for the substrates or on the KA values for the divalent cation cofactors. The predominant factor in dictating the catalytic properties of calcineurin is the divalent cation cofactor. For example, with Mg2+ as a cofactor, the phosphatase exhibits an optimum around pH 8.0-8.5; while with a transition metal ion as a cofactor, the optimum is around pH 7.0-7.5, regardless of whether Thr(P)-inhibitor-1 or Ser(P)-casein serves as a substrate, in the absence or the presence of Ca2+ X calmodulin.     

Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum.

A protein kinase which phosphorylated histone and protamine was partially purified from bovine cerebellum. Casein and phosvitin were inert as substrates. The enzyme did not require any cyclic nucleotide. A sulfhydryl compound such as 2-mercaptoethanol, glutathione, or cysteine was necessary for the reaction. The optimum pH was 8.5 to 9.0 Km values for ATP and whole histone were 3.3 X 10(-6) M and 150 microgram/ml, respectively. The optimum concentration of Mg2+ varied with histone fractions employed; with H2B histone as substrate the enzyme was most active at 50 to 100 nM Mg2", whereas with H1 and H2A histones the maximum activity was observed at 5 to 10 mM Mg2+ and with H3 and H4 histones the enzyme was active over a range of 5 to 75 mM Mg2+. The enzyme phosphorylated Ser-32 and Ser-36 in H2B histone and Ser-38 in H1 histone, although the reaction with Ser-36 in H2B histone was very slow. The molecular weight was 6.4 X 10(4). The sedimentation coefficient and Stokes radium were about 4.5 and 29 A, respectively. The enzyme showed heterogeneity upon isoelectrofocusing electrophoresis with isoelectric points of 5.6, 6.0, and 6.6. The enzyme was not inhibited by protein inhibitor nor by the regulatory subunit of cyclic AMP-dependent protein kinase. Preliminary analysis suggested that the enzyme was produced from its precursor protein by a limited proteolytic reaction.     

Distinct metal cofactor-induced conformational states in the NAD-specific malic enzyme of Escherichia coli as revealed by proteolysis studies

Evidence is presented for the existence of altered ligand-stabilized conformational states of the NAD-specific malic enzyme (L-malate:NAD+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.38), of Escherichia coli in the presence of Mg2+ and Mn2+, as identified by their susceptibilities to proteolysis. The rate of tryptic digestion of the enzyme is significantly decreased in the Mg2+-form of the enzyme when the product, NADH, or the allosteric effectors, coenzyme A and aspartate, are present in the digestion mixture. In contrast, little difference in the rate of tryptic digestion is observed in the degree of protection of the enzyme by the two metal cofactors, either alone, or in the presence of the substrates, malate and NAD. The results are consistent with the previously proposed hypothesis of Milne and Cook (Biochemistry 18, (1979) 3604-3610) that Mg2+ and Mn2+ stabilize two distinct conformational states of the enzyme. The results are discussed in relation to the altered kinetic response of the enzyme to substrates and effectors in the presence of the two metal cofactors.     

Enzymes of the last steps of chlorophyll biosynthesis: modification of the substrate structure helps to understand the topology of the active centers

Enzymes catalyzing two of the late steps of chlorophyll biosynthesis are NADPH:protochlorophyllide oxidoreductase (POR), responsible for the light-dependent reduction of protochlorophyllide to chlorophyllide, and chlorophyll synthase that catalyses the esterification of chlorophyllide to chlorophyll. Inhibitors of these enzymes are of interest as potential herbicides. Both enzymes presumably form a complex, and the question arose whether chlorophyll synthase can react with chlorophyllide while it is still bound to POR. Here, we describe the chemical modification of protochlorophyllides and chlorophyllides with space-filling substituents at rings A, B, and E of the tetrapyrrole macrocycle and the reactivity of the modified substrates. Both enzymes tolerate the large and flexible phenylamino substituent at ring B, indicating that ring B points toward the enzyme surface while the substrate is bound. On the basis of the standard compound zinc protopheophorbide a (100% activity), the 7(1)-phenylamino derivative shows a comparable activity (83%) with POR that is higher than that of the parent formyl derivative zinc protopheophorbide b (58% activity). In contrast, the 3(1)-phenylamino derivative is less active (12%) than the parent formyl compound zinc protopheophorbide d (49% activity), indicating that the binding pocket leaves less space around ring A than around ring B. Almost no space must be left around ring E because substitution of the 13(2)-carboxymethyl ester (100% activity) by the 13(2)-carboxyethyl ester reduces the activity to 0.2%. Chlorophyll synthase leaves somewhat more space around ring E on the A side of the tetrapyrrole in the binding pocket; substitution of the 13(2)-proton (100% activity) by a methoxy group (53% activity) and an ethoxy group (11% activity) is tolerated to a certain extent, while the carbomethoxy group in this position is not accepted. Opening of ring E to a chlorin e6 dimethylester is tolerated (39% activity), while the large benzylamide residue at this site leads to the loss of activity. We conclude that the tetrapyrroles bind to both enzymes in the same direction: rings C, D, and E are oriented to the interior of the binding cleft, and rings A and B are oriented to the surface of the enzyme; this excludes simultaneous binding to both enzymes.