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J Steyaert

Publications and source records attributed to J Steyaert.

At least 55 records · Page 3Linked to original sources
PubMed1995

A two-binding-site kinetic model for the ribonuclease-T1-catalysed transesterification of dinucleoside phosphate substrates.

Ribonucleases have been found to have subsites that confer large rate enhancements but do not contribute to substrate binding. In this study, we present a kinetic model that formally explains how subsite binding energy is converted into chemical activation energy. The proposed mechanism takes into account a primary specificity site and a subsite, both of which must be occupied for chemical turnover. An unstable reaction intermediate is formed upon binding of the polymeric substrate monomers at the corresponding subsites. The structure of this reaction intermediate resembles the transition state of the catalysed transphosphorylation reaction. Similar mechanisms may be used by other depolymerizing enzymes including nucleases, glycosidases, and proteases.

Binding Sites↗
PubMed1995

Primary structure and partial characterization of a life-cycle-regulated cysteine protease from Trypanosoma (Nannomonas) congolense.

Trypanosoma (Nannomonas) congolense is an important pathogenic parasite of domestic livestock in Africa. We have cloned a cDNA encoding a prepro-cysteine protease of this protozoan, the sequence of which indicates it is an early mRNA processing intermediate. Northern analysis demonstrates a life-cycle-stage specificity similar to previously described enzymatic data. The deduced amino-acid sequence shows extensive similarity to cysteine proteases of other parasitic protozoa, as well as papain and cathepsin L. As with other African trypanosomes, a poly-proline tract connects the catalytic domain with an unusual C-terminal extension.

Amino Acid Sequence↗
PubMed1995

Neurofibromatosis type 1 in childhood: correlation of MRI findings with intelligence.

In a group of 28 children with neurofibromatosis type 1 aged between 4 and 16 years, neuroradiological findings were correlated with intelligence as measured by the Wechsler scales. The presence or specific location in the brain of T2 weighted prolonged signals on MRI was not associated with cognitive problems. No other physical characteristics associated with neurofibromatosis type 1 were found to correlate significantly with IQ. At the present these T2 weighted hyper-intense spots should not be used to predict neurofibromatosis type 1 associated cognitive problems.

Analysis of Variance↗
PubMed1994

Personality profile in adult female fragile X carriers: assessed with the Minnesota Multiphasic Personality Profile (MMPI).

To assess if the higher incidence of psychiatric morbidity in fragile X carriers is related to a particular pathological personality profile, we obtained a Minnesota Multiphasic Personality Profile from 11 normally intelligent (female) fragile X carriers. The sample mean for the clinical and validity scales all fell within the normal range. Although no pathological profile was found, some unexpected results emerged: low scores for the scales "schizophrenia" and "social introversion" and a so-called faking-good profile on the validity scales. This combination suggests that the subjects might not be aware of some personality characteristics in these areas. Implications for further research are discussed.

Adult↗
PubMed1994

Apparently enhanced visual information processing in female fragile X carriers: preliminary findings.

Eighteen normally intelligent adult female fragile X carriers were tested with the "de Sonneville Visual Attention Tasks," a computer-based set of reaction-time experiments, in order to assess different aspects of their attention capacity. The subject group was compared with a group of 48 adults, who were not known to be fragile X carriers. We publish preliminary results of this study. These show that the fragile X carriers have significantly faster reaction times at a substantial number of the tests. This suggests that normally intelligent female fragile X carriers might process simple visual information, such as the targets offered in these reaction time experiments, at a faster rate than control women.

Adult↗
PubMed1994

Crystallographic study of Glu58Ala RNase T1 x 2'-guanosine monophosphate at 1.9-A resolution.

Glu58 is known to participate in phosphodiester transesterification catalyzed by the enzyme RNase T1. For Glu58 RNase T1, an altered mechanism has been proposed in which His40 replaces Glu58 as the base catalyst [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. Glu58Ala Rnase T1 has been cocrystallized with guanosine 2'-monophosphate (2'-GMP). The crystals are of space group P2(1), with one molecule per asymmetric unit (a = 32.44 A, b = 49.64 A, c = 26.09 A, beta = 99.17 degrees). The three-dimensional structure of the enzyme was determined to a nominal resolution of 1.9 A, yielding a crystallographic R factor of 0.178 for all X-ray data. Comparison of this structure with wild-type structures leads to the following conclusions. The minor changes apparent in the tertiary structure can be explained by either the mutation of Glu58 or by the change in the space group. In the active site, the extra space available through the mutation of Glu58 is occupied by the phosphate group (after a reorientation) and by a solvent molecule replacing a carboxylate oxygen of Glu58. This solvent molecule is a candidate for participation in the altered mechanism of this mutant enzyme. Following up on a study of conserved water sites in RNase T1 crystallized in space group P2(1)2(1)2(1) [Malin, R., Zielenkiewicz, P., & Saenger, W. (1991) J. Mol. Biol. 266, 4848-4852], we investigated the hydration structure for four different packing modes of RNase T1.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1994

Investigation of the functional interplay between the primary site and the subsite of RNase T1: kinetic analysis of single and multiple mutants for modified substrates.

We report on the functional cooperativity of the primary site and the subsite of ribonuclease T1 (RNase T1; EC 3.1.27.3). The kinetic properties of the single Tyr-38-Phe and Asn-98-Ala mutants have been compared with those of the corresponding double mutant. The Tyr-38-Phe mutation has been used to probe enzyme-substrate interactions at the primary site; the Asn-98-Ala mutation monitors subsite interactions. In addition to the dinucleoside phosphate substrate GpC, we measured the kinetics for GpMe, a synthetic substrate in which the leaving nucleoside cytosine has been replaced by methanol. All data were combined in a triple mutant box to analyze the interplay between Tyr-38, Asn-98, and the leaving group. The free energy barriers to kcat, introduced by the single Tyr-38-Phe and Asn-98-Ala mutations are not additive in the corresponding double mutant. The energetic coupling between both mutations is independent of the binding of the leaving cytosine at the subsite. We conclude that the coupling of the Tyr-38-Phe and Asn-98-Ala mutations arises through distortion or reorientation of the 3'-guanylic acid moiety bound at the primary site. The experimental data indicate that the enzyme-substrate interactions beyond the scissile phosphodiester bond contribute to catalysis through the formation of new or improved contacts in going from ground state to transition state, which are functionally independent of primary site interactions.

Amino Acids↗
PubMed1993

The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase.

DNA topoisomerases perform essential roles in DNA replication, gene transcription, and chromosome segregation. Recently, we identified a new type of topoisomerase II poison: the CcdB protein of plasmid F. When its action is not prevented by CcdA protein, the CcdB protein is a potent cytotoxin. In this paper, using purified CcdB, CcdA and gyrase, we show that CcdB protein efficiently traps gyrase in a cleavable complex. The CcdA protein not only prevents the gyrase poisoning activity of CcdB but also reverses its effect on gyrase. The mechanism by which the CcdB protein induces DNA strand breakage is closely related to the action of quinolone antibiotics. However, the ATP dependence of the CcdB cleavage process differentiates the CcdB mechanism from quinolone-dependent reactions because the quinolone antibiotics stimulate efficient DNA breakage, whether or not ATP is present. We previously showed that bacteria resistant to quinolone antibiotics are sensitive to CcdB and vice versa. Elucidation of the mechanism of action of CcdB protein may permit the design of drugs targeting gyrase so as to take advantage of this new poisoning mechanism.

Adenosine Triphosphate↗
PubMed1993

Crystal structure determination and refinement at 2.3-A resolution of the lentil lectin.

We report on the X-ray structure determination of the orthorhombic crystal form of lentil lectin by molecular replacement using the pea lectin coordinates as a starting model. The structure was refined at 2.3-A resolution with a combination of molecular dynamics refinement and classical restrained least-squares refinement. The final R value for all data Fo > 1 sigma (Fo) between 7.0- and 2.3-A resolution is 0.164%, and deviations from ideal bond distances are 0.014 A. The C-terminus of the beta-chain proved to be 23 amino acids longer than found in previous studies. This together with several inconsistencies between the previously determined amino acid sequence and the observed electron density forced a redetermination of the amino acid sequence of the protein. The overall structure is very similar to that of pea lectin and isolectin I of Lathyrus ochrus, the most prominent deviations being confined to loop regions and the regions of intermolecular contact. The largest difference between the pea and lentil lectin monomers is situated in the loop region of amino acids 73-79 of the beta chain. There are no significant differences between the two crystallographic independent lentil lectin monomers in the asymmetric unit. The model includes 104 well-defined water molecules, of which a significant number have a counterpart in the pea lectin structure. As for the other legume lectins, each lentil lectin monomer contains one calcium ion in a highly conserved environment. On the contrary, the manganese binding sites are distorted with respect to the pea lectin and concanavalin A structures. The Asp beta 121 side chain apparently does not ligate the Mn2+ ion. This difference is consistent in both lentil lectin monomers and agrees with earlier solution studies. Possible implications for oligosaccharide binding are discussed.

Amino Acid Sequence↗
PubMed1993

Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein.

Large crystals of the Escherichia coli F plasmid CcdB killer protein were grown from solutions containing 32% ammonium sulphate. The crystals belong to space group P4(2)2(1)2 with a = b = 104.52 A and c = 88.45 A or P2(1)2(1)2(1) with a = 77.62 A, b = 93.28 A and c = 141.44 A. Both crystal forms diffract to 2.6 A resolution. Structure determination by multiple isomorphous replacement is under way.

Bacterial Proteins↗
PubMed1993

Functional interactions among the His40, Glu58 and His92 catalysts of ribonuclease T1 as studied by double and triple mutants.

We report on the functional interplay between the His40, Glu58 and His92 catalysts of ribonuclease T1. The kinetic properties of the single His40Ala, Glu58Ala and His92Gln mutants have been compared with those of the corresponding double and triple mutants. When His40, Glu58 and His92 are mutated separately or together, we observe large effects on turnover but only minor effects on substrate binding. The free energy barriers to kcat introduced by the single His40Ala, Glu58Ala and His92Gln mutations are non-additive in the corresponding His40Ala + Glu58Ala, Glu58Ala + His92Gln and His40Ala + His92Gln double mutants; a significant dependence of the pairwise interactions on the third residue has been observed. Using a pair of related triple mutant boxes, we were able to divide the apparent coupling energies as calculated from the kinetic parameters of the various mutants either quantitatively or qualitatively into terms that measure intrinsic interaction energies between His40, Glu58 and His92 in wild-type enzyme and terms that account for a change in reaction mechanism that is associated with the Glu58Ala single mutation. In most cases, non-additivity may be explained by changes in enzyme mechanism. Apart from this change in mechanism, functional interactions have to be considered between His40 and Glu58, Glu58 and His92 as well as between His40 and His92 to explain non-additivity in all double-mutant cycles. It is concluded that the collaborative effects of His40, Glu58 and His92 decrease the energetic barrier to kcat by 6.8 kcal/mol. The overall effect caused by the triple mutation is smaller than that expected from the product of the fractional kcat values resulting from the individual mutations (11.0 kcal/mol), illustrating the limitations of using single mutants to probe the energetics of a catalytic group whose function is dependent upon interactions with others.

Catalysis↗
PubMed1992

Role of histidine-40 in ribonuclease T1 catalysis: three-dimensionalstructures of the partially active His40Lys mutant.

Histidine-40 is known to participate in phosphodiester transesterification catalyzed by the enzyme ribonuclease T1. A mutant enzyme with a lysine replacing the histidine-40 (His40Lys RNase T1) retains considerable catalytic activity [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. We report on the crystal structures of His40Lys RNase T1 containing a phosphate anion and a guanosine 2'-phosphate inhibitor in the active site, respectively. Similar to previously described structures, the phosphate-containing crystals are of space group P2(1)2(1)2(1), with one molecule per asymmetric unit (a = 48.27 A, b = 46.50 A, c = 41.14 A). The complex with 2'-GMP crystallized in the lower symmetry space group P2(1), with two molecules per asymmetric unit (a = 49.20 A, b = 48.19 A, c = 40.16 A, beta = 90.26). The crystal structures have been solved at 1.8- and 2.0-A resolution yielding R values of 14.5% and 16.0%, respectively. Comparison of these His40Lys structures with the corresponding wild-type structures, containing 2'-GMP [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368] and vanadate [Kostrewa, D., Hui-Woog Choe, Heinemann, U., & Saenger, W. (1989) Biochemistry 28, 7692-7600] in the active site, respectively, leads to the following conclusions. First, the His40Lys mutation causes no significant changes in the overall structure of RNase T1; second, the Lys40 side chains in the mutant structures occupy roughly the same space as His40 in the corresponding wild-type RNase T1 structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.

Alanine↗
PubMed1991

Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N.

We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U. The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters. Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme. The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks). Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU. This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1991

Subsite interactions of ribonuclease T1: Asn36 and Asn98 accelerate GpN transesterification through interactions with the leaving nucleoside N.

We previously presented evidence that ribonuclease T1 (RNase T1; EC 3.1.27.3) contains a subsite that, by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis. The kcat values for transphosphorylation follow the order GpC greater than GpA greater than GpU whereas the equilibrium dissociation constants for these substrates are very similar [Steyaert, J., Wyns, L., & Stanssens, P. (1991) Biochemistry (preceding paper in this issue)]. Consistent with this notion, we find that the rate of transesterification of the synthetic substrate GpMe, in which the leaving nucleoside is replaced by a methanol group, is at least 3 orders of magnitude lower than that of GpN substrates. The enzyme's affinity for GpMe is very similar to that for the various GpN substrates, indicating that the apparent contribution of the leaving nucleoside to ground-state binding is minimal. To identify the side chains that belong to the RNase T1 subsite, we searched for amino acid substitutions that differentially affect the transesterification kinetics of GpNs versus GpMe. The Asn36Ala, Tyr38Phe, His92Gln, and Asn98Ala mutants have been analyzed. Of these, the Asn36Ala and Asn98Ala substitutions reduce the transphosphorylation rate of the different GpNs considerably whereas they have virtually no effect on the rate of GpMe transphosphorylation. This observation shows that the Asn36 and Asn98 amide functions are part of the RNase T1 subsite. The sum of the contributions of the two residues accounts quite precisely for the differences in turnover rates among GpC, GpA, and GpU.

Amino Acid Sequence↗
PubMed1991

Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1.

In the crystal structure of the ribonuclease T1 (RNase T1; EC 3.1.27.3)-2'-GMP complex the hydrogen-bonding potential of the guanine base is saturated [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368]. The oxygens of the Glu46 carboxylate and the Asn98 main-chain carbonyl act as hydrogen-bond acceptors for the N(1)H-C(2)-N(2)H2 part of the base. We measured the transesterification kinetics of wild-type and Glu46Ala RNase T1 using the GpU, IpU, and XpU series of analogous substrates. We found that the N(1)H---Glu46 O epsilon 1, the N(2)H---Glu46 O epsilon 2, and the N(2)H---Asn98 O hydrogen bonds have an apparent contribution of 2.7, 1.1, and 1.2 kcal/mol to the interaction energy of the enzyme and the transition state of the substrate. Wild-type RNase T1 discriminates guanine from nonionized xanthine (a guanine analogue in which the exocyclic amino group is replaced by an oxygen) by about 4.4 kcal/mol. Loss of the specific hydrogen bonds with the exocyclic amino group of the guanine base accounts for 2.4 kcal/mol of this discrimination energy; 2.0 kcal/mol is due to unfavorable non-H-bonded oxygen-oxygen contacts in the enzyme-xanthine complex. A pH dependence study shows that the deprotonated form of xanthine (i.e., the 6-keto-2-enolate anion; pKa = 5.4) is far less preferred, if not excluded, as substrate by wild-type RNase T1; this may be attributed to an electrostatic repulsion of the negatively charged xanthine by the Glu46 carboxylate group.

Asparagine↗
PubMed1990

Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine.

Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.

Alanine↗