Background-Q waves on a 12-lead ECG are markers of a prior myocardial infarction (MI). However, they may regress or even disappear over time, and there is no specific ECG sign of a non-Q-wave MI. Fragmented QRS complexes (fQRSs), which include various RSRЈ patterns, without a typical bundle-branch block are markers of altered ventricular depolarization owing to a prior myocardial scar. We postulated that the presence of an fQRS might improve the ability to detect a prior MI compared with Q waves alone by ECG. Methods and Results-A cohort of 479 consecutive patients (meanϮSD age, 58.2Ϯ13.2 years; 283 males) who were referred for nuclear stress tests was studied. The fQRS included various morphologies of the QRS (Ͻ120 ms), which included an additional R wave (RЈ) or notching in the nadir of the S wave, or Ͼ1 RЈ (fragmentation) in 2 contiguous leads, corresponding to a major coronary artery territory. The Q wave was present in 71 (14.8%) patients, an fQRS was present in 191 (34.9%) patients, and an fQRS and/or a Q wave was present in 203 (42.3%) patients. Sensitivity, specificity, and the negative predictive value for myocardial scar as detected by single photon emission computed tomography analysis were 36.3%, 99.2%, and 70.8%, respectively, for the Q wave alone; 85.6%, 89%, and 92.7%, respectively, for the fQRS; and 91.4%, 89%, and 94.2%, respectively, for the Q wave and/or fQRS. Conclusions-The fQRS on a 12-lead ECG is a marker of a prior MI, defined by regional perfusion abnormalities, which has a substantially higher sensitivity and negative predictive value compared with the Q wave.

The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (؊4 to ؉4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism,where the RNA polymerase forms an initial weak ED a complex rapidly with an equilibrium association constant K 1 . The ED a complex then undergoes a conformational change to ED b , wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of ED a to ED b is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, ⌽10 and ⌽3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, k on (K 1 k 2 ), and the dissociation rate constant, k off (k ؊2 ), and thermodynamics, the equilibrium constants of the two steps (K 1 and k 2 /k ؊2 ) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower k on , the 4-fold higher k off , and the 2-5-fold weaker equilibrium interactions together make ⌽3.8 a weaker promoter relative to ⌽10.Bacteriophage T7 RNA polymerase is a 98-kDa single subunit polymerase that catalyzes synthesis of RNA complementary in sequence to the template DNA (1). The phage enzymes are among the simplest RNA polymerases known, as no accessory proteins are necessary for specific initiation, elongation, or termination of transcription (2, 3). The 17 promoters of bacteriophage T7 direct specific initiation of RNA synthesis that occurs in a rapid and processive manner (4, 5). Due to their simplicity these enzymes serve as model systems to understand, in depth, the mechanisms of transcription initiation, elongation, or termination.Initiation of transcription occurs by recognition and binding of the RNA polymerase to a promoter DNA sequence. This event is recognized as one of the important steps at which transcription and gene expression is regulated. The 17 bacteriophage T7 promoters share consensus sequence from Ϫ17 to ϩ6 position relative to the transcription start site at ϩ1 (6). The class III gene promoters of T7 are absolutely conserved in DNA sequence, whereas the class II gene promoters diff...

The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

To probe the role of the Asp-99. . .His-48 pair in phospholipase A, (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99 + Asn-99 (D99N) of bovine pancreatic PLAZ was undertaken. Crystals of D99N belong to the trigonal space group P3121 and were isomorphous to the wild type (WT) (Noel J P et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH, group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH, group of Asn-99. The NH, group now forms a direct hydrogen bond with the carbonyl group of Ala-I.Keywords: histidine tautomeric form; missing structural water; PLA2 D99N mutant; structure-function relationship; X-ray structure Phospholipase A2 (Fig. 1A) hydrolyzes the sn-2 ester bond of phospholipids. The studies on the mechanism of action of PLA2 have generated immense pharmacological interest. The mechanism involves binding of the enzyme to the lipid-water interface, productive binding of a single lipid molecule in the active site followed by hydrolysis (Scott et al., 1990). The interfacial binding site is essentially at the surface and includes the residues of the N-terminal helix-A, the calcium ion binding loop, and the loop connecting helix-D and the 0-sheet (Dijkstra et al., 1981; see Kinemage 1). The catalytic triad Asp-99, His-48, and the waReprint requests to: Muttaiya Sundaralingam or Ming-Daw Tsai, Department