Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate.

NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements.

Immediate effect of aortic valve replacement for aortic stenosis on left ventricular diastolic chamber stiffness.

Diastolic dysfunction is common after coronary artery bypass surgery, and we hypothesized that left ventricular (LV) hypertrophy associated with aortic stenosis may lead to worsening LV diastolic function after aortic valve replacement for aortic stenosis. Transesophageal echocardiographic LV images and simultaneous pulmonary arterial wedge pressures were used to define the LV diastolic pressure cross-sectional area relation before and immediately after aortic valve replacement for aortic stenosis in 14 patients. In all patients, LV diastolic chamber stiffness increased, as evidenced by a leftward shift in the LV diastolic pressure cross-sectional area relation. At comparable LV filling (pulmonary arterial wedge) pressures the mean LV end-diastolic cross-sectional area preoperatively was 17.9 +/- 1.7 cm2, but decreased by 32% after aortic valve replacement to 12.1 +/- 1.2 cm2 (p = 0.0001). In conclusion, after aortic valve replacement, diastolic chamber stiffness increased in all patients.

Characterization of a buried neutral histidine in Bacillus circulans xylanase: internal dynamics and interaction with a bound water molecule.

NMR spectroscopy was used to characterize the dynamic behavior of His149 in Bacillus circulans xylanase (BCX) and its interaction with an internal water molecule. Rate constants for the specific acid- and base-catalyzed exchange following bimolecular kinetics (EX2) of the nitrogen-bonded H epsilon 2 of this buried, neutral histidine were determined. At pDmin 7.0 and 30 degrees C, the lifetime for this proton is 9.9 h, corresponding to a protection factor of approximately 10(7) relative to that predicted for an exposed histidine. The apparent activation energies measured for specific acid and base catalysis (7.0 and 17.4 kcal/mol) indicate that exchange occurs via local structural fluctuations. Consistent with its buried environment, the N epsilon 2-H bond vector of His149 shows restricted mobility, as evidenced by an order parameter S2 = 0.83 determined from 15N relaxation measurements. The crystal structure of BCX reveals that a conserved, buried water hydrogen-bonds to the H epsilon 2 of His149. Strong support for this interaction in solution is provided by the observation of a negative nuclear Overhauser effect (NOE) and positive rotating-frame Overhauser effect (ROE) between His149 H epsilon 2 and a water molecule with the same chemical shift as the bulk solvent. However, the chemical shift of H epsilon 2 (12.2 ppm) and a D/H fractionation factor close to unity (0.89 +/- 0.02) indicate that this is not a so-called low-barrier hydrogen bond. Lower and upper bounds on the lifetime of the internal water are estimated to be 10(-8) and 10(-3) s. Therefore the chemical exchange of solvent protons with those of His149 H epsilon 2 and the diffusion or physical exchange of the internal water to which the histidine is hydrogen-bonded differ in rate by over 7 orders of magnitude.

Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.

N-acetylaspartate complexes with calcium and lanthanide ions.

N-acetylaspartate (NAA) is the one of the most prominent resonances observed in the solvent-suppressed NMR spectrum of the human brain. Although it is present in the brain at about 10 mM, its precise metabolic function is still unclear, We have examined the NAA as a potential chelator for divalent metal ions such as Ca2+. We have employed the perturbations induced by Ln3+ ions in the 1H and 13C NMR spectrum of NAA to monitor formation of NAA complexes. 1H NMR measurements showed that the dissociation constants for the formation of Eu(3+)-NAA, Yb(3+)-NAA, and Ca(2+)-NAA complexes were 0.07, 0.13, and 0.86 mM, respectively. Scatchard analysis of the results indicates the formation of a 1:1 metal-ligand complex. We also inferred the structure of the NAA-metal ion complex from an analysis of paramagnetic perturbations induced in the 1H NMR and 13C NMR spectra of NAA. The structural analysis of the NAA-metal ion complex indicates that the two carboxylic groups participate in chelating the metal ion, forming the binding site for the metal ion.

Ischemic papillary muscle dysfunction in an adult with a parachute mitral valve.

A case of severe mitral regurgitation secondary to an infarct of a single papillary muscle in a 65-year-old female with a parachute mitral valve is described. To the best of our knowledge this is the oldest patient reported with this rare congenital anomaly. The clinical and echocardiographic features are discussed.

Right coronary artery stenosis: an independent predictor of atrial fibrillation after coronary artery bypass surgery.

OBJECTIVES: This study attempted to determine the importance of severe proximal right coronary artery disease as a predictor of atrial fibrillation in patients after coronary artery bypass surgery. BACKGROUND: Studies in patients undergoing noncardiac surgery have suggested that ischemia in the right coronary artery distribution is associated with a high incidence of atrial fibrillation. However, the importance of right coronary artery disease as a predictor of atrial fibrillation after bypass surgery is unknown. METHODS: The occurrence of sustained postoperative atrial fibrillation was studied prospectively in 168 consecutive patients undergoing coronary artery bypass grafting. Patients were followed up postoperatively until discharge. Severe right coronary artery stenosis was defined as > or = 70% lumen narrowing. RESULTS: Of 104 patients with proximal or mid right coronary artery stenosis, 45 (43%) had atrial fibrillation postoperatively compared with 12 (19%) of the 64 patients without significant right coronary disease (p = 0.001). Univariate predictors of atrial fibrillation included right coronary artery stenosis (p = 0.001), advancing age (p = 0.0001) and lack of beta-adrenergic blocking agent therapy after bypass surgery (p = 0.0004). Multivariate adjusted risk of developing atrial fibrillation after bypass surgery increased with the presence of severe right coronary artery disease (odds ratio 3.69, 95% confidence interval [CI] 1.61 to 8.48), advancing age (odds ratio 2.24/10 years, CI 1.48 to 3.41) and male gender (odds ratio 2.36, CI 1.01 to 5.49). The use of beta-blockers postoperatively was associated with a protective effect (odds ratio 0.4, CI 0.17 to 0.80). CONCLUSIONS: The presence of severe right coronary artery stenosis is an independent and powerful predictor of atrial fibrillation after coronary artery bypass surgery. In association with age, gender and postoperative beta-blocker therapy, these variables can be used to identify patients at increased risk for developing this arrhythmia.

Increased left ventricular diastolic chamber stiffness immediately after coronary artery bypass surgery.

OBJECTIVES: The aim of this study was to assess the incidence and severity of left ventricular diastolic dysfunction immediately after coronary artery bypass surgery by utilizing simultaneous transesophageal echocardiographic and hemodynamic monitoring. BACKGROUND: Left ventricular diastolic dysfunction has been documented after coronary bypass surgery, but its measurement has been technically difficult to acquire and limited by dependence on loading conditions. METHODS: End-diastolic pressure-area curves were constructed before and immediately after coronary bypass surgery in 20 patients. Transesophageal echocardiographic images at the midpapillary level of the left ventricle and hemodynamic data were recorded. Volume status was manipulated to alter loading conditions, and multiple measurements were taken at each loading condition. RESULTS: Diastolic function worsened in all patients, as manifested by a postoperative leftward shift of the end-diastolic pressure-area curve. At a comparable preload, mean end-diastolic area +/- SEM decreased by 15% from 17.6 +/- 0.8 to 14.9 +/- 0.8 cm2 postoperatively (p = 0.0001). CONCLUSIONS: Left ventricular diastolic chamber stiffness frequently increases immediately after coronary artery bypass surgery. Simultaneous hemodynamic and transesophageal echocardiographic monitoring, through the construction of end-diastolic pressure-area curves, is a useful method to evaluate diastolic function and guide management after cardiac surgery.

Left ventricular dysfunction during infrarenal abdominal aortic aneurysm repair.

BACKGROUND: Clinical observations suggest that pulmonary artery occlusion pressure (PAOP) underestimates the resuscitative volumes required prior to release of aortic cross-clamp. METHODS: To investigate pressure-volume relationships associated with repair of abdominal aortic aneurysm (AAA), we simultaneously monitored PAOP by pulmonary artery catheter (PAC) and estimated left ventricular (LV) diastolic volume using two-dimensional transesophageal echocardiography (TEE) in 22 patients undergoing AAA repair. Data from PAC monitoring and TEE were collected before, during, and after aortic occlusion. TEE cross-sectional images were obtained at the mid-papillary level. RESULTS: Overall, PAOP correlated with left ventricular end-diastolic area (LVEDA), but the correlation was not particularly strong (r = 0.37, P < 0.0001). Even within individual patients, LVEDA varied widely for a given PAOP. The strength of the correlation between PAOP and LVEDA also appeared to deteriorate during the course of surgery. The best correlation was seen prior to aortic cross-clamping (r = 0.50, P < 0.0001), but fell somewhat during aortic cross-clamping (r = 0.41, P < 0.0001), and even further after unclamping (r = 0.25, P = 0.005). CONCLUSION: This study demonstrates a relatively weak correlation between PAOP and LVEDA using intraoperative TEE during AAA repair. Furthermore, the strength of the correlation worsened during surgery, particularly after unclamping. Although unclear at this time, this finding may be attributable to changes in LV compliance. We found TEE to be a valuable adjunct in guiding volume resuscitation of patients undergoing AAA repair.

Systolic anterior motion of the mitral valve after valve repair without an annular ring.

A 78-year-old woman underwent mitral reconstruction, consisting of a quadrangular posterior leaflet resection without a concomitant annular ring, for symptomatic mitral regurgitation. Postoperatively she became hypotensive while being treated with dopamine. Transesophageal echocardiography revealed systolic anterior motion of the mitral valve with significant mitral regurgitation. With discontinuation of the dopamine regimen, institution of phenylephrine administration, and volume loading of the ventricle the systolic anterior motion disappeared as did the mitral regurgitation. The patient recovered uneventfully.

Isotope effects in peptide group hydrogen exchange.

Kinetic and equilibrium isotope effects in peptide group hydrogen exchange reactions were evaluated. Unlike many other reactions, kinetic isotope effects in amide hydrogen exchange are small because exchange pathways are not limited by bond-breaking steps. Rate constants for the acid-catalyzed exchange of peptide group NH, ND, and NT in H2O are essentially identical, but a solvent isotope effect doubles the rate in D2O. Rate constants for base-catalyzed exchange in H2O decrease slowly in the order NH > ND > NT. The alkaline rate constant in D2O is very close to that in H2O when account is taken of the glass electrode pH artifact and the difference in solvent ionization constant. Small equilibrium isotope effects lead to an excess equilibrium accumulation of the heavier isotopes by the peptide group. Results obtained are expressed in terms of rate constants for the random coil polypeptide, poly-DL-alanine, to provide reference rates for protein hydrogen exchange studies as described in Bai et al. [preceding paper in this issued].

Effects of isometric exercise on the end-diastolic pressure, volumes, and function of the left ventricle in man.

Changes induced in left ventricular (LV) hemodynamics by isometric exercise were analyzed in 43 patients: 30 with coronary heart disease (CAD), four with noncoronary heart disease, nine normal. Volumes were angiographically determined and correlated with left ventricular end-diastolic pressure (LVEDP) both at rest and during the fifth minute of 30% sustained handgrip (HNG). All normals and eight with CAD improved LV function during HNG. LVEDP decreased or remained constant, end-diastolic volume (EDV) decreased, end-systolic volume (ESV) decreased, as ejection fraction (EF) remained constant. None of these eight CAD cases altered their regional LV contraction pattern during HNG. Twenty-five patients, 21 CAD and four nonCAD, showed diminished LV function during HNG. LVEDP increased, EDV decreased, ESV increased, as EF declined. In these 21 CAD patients, at least one major coronary vessel was narrowed 70% or more and, with but two exceptions, was not supported by adequate collaterals. In 18, new asynergic zones developed in previously normally contracting areas or pre-existing asynergic zones extended during HNG.