Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope.

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

The influence of diabetes on postoperative complications following colorectal surgery.

BACKGROUND: Diabetes mellitus has been commonly associated with poor surgical outcomes. The aim of this meta-analysis was to assess the impact of diabetes on postoperative complications following colorectal surgery. METHODS: Medline, Embase and China National Knowledge Infrastructure electronic databases were reviewed from inception until May 9th 2020. Meta-analysis of proportions and comparative meta-analysis were conducted. Studies that involved patients with diabetes mellitus having colorectal surgery, with the inclusion of patients without a history of diabetes as a control, were selected. The outcomes measured were postoperative complications. RESULTS: Fifty-five studies with a total of 666,886 patients comprising 93,173 patients with diabetes and 573,713 patients without diabetes were included. Anastomotic leak (OR 2.407; 95% CI 1.837-3.155; p < 0.001), surgical site infections (OR 1.979; 95% CI 1.636-2.394; p < 0.001), urinary complications (OR 1.687; 95% CI 1.210-2.353; p = 0.002), and hospital readmissions (OR 1.406; 95% CI 1.349-1.466; p < 0.001) were found to be significantly higher amongst patients with diabetes following colorectal surgery. The incidence of septicemia, intra-abdominal infections, mechanical failure of wound healing comprising wound dehiscence and disruption, pulmonary complications, reoperation, and 30-day mortality were not significantly increased. CONCLUSIONS: This meta-analysis and systematic review found a higher incidence of postoperative complications including anastomotic leaks and a higher re-admission rate. Risk profiling for diabetes prior to surgery and perioperative optimization for patients with diabetes is critical to improve surgical outcomes.

Colorectal resection via natural orifice specimen extraction versus conventional laparoscopic extraction: a meta-analysis with meta-regression.

BACKGROUND: Studies have shown differences in postoperative outcomes between two minimally invasive extraction methods for colorectal lesions-natural orifice specimen extraction surgery (NOSES) and conventional laparoscopic surgery (CLS). The aim of this study was to discover the major differences in NOSES and CLS to refine current practice. METHODS: Electronic databases were searched for articles comparing NOSES and CLS from inception till March 2020. Weighted mean differences (WMD) and odds ratio (OR) were estimated for continuous and dichotomous outcomes, respectively. Summary statistics were calculated using the DerSimonian and Laird random effects. RESULTS: Twenty-one studies (15 on malignant disease, 4 on benign disease, 2 on both) were included in this meta-analysis, totalling 2378 patients (1079 NOSE, 1299 CLS). NOSE was associated with decreased: intraoperative bleeding (WMD: - 10.652 ml; 95% CI: - 18.818 ml to - 2.482 ml; p < 0.001), pain score (WMD: - 1.520; 95% CI - 1.965 to - 1.076; p < 0.001), time to flatus (WMD: - 0.306 days; 95% CI: - 0.526 to - 0.085 days; p < 0.001), length of hospital stay (WMD: - 1.048 days; 95% CI: - 1.488 to - 0.609 days; p < 0.001), and total morbidity (OR: 0.548; 95% CI: 0.387 to 0.777; p = 0.001). Subgroup analyses showed significant differences between malignant and benign lesions for intraoperative bleeding (p = 0.011) and pain score (p = 0.010). Meta-regression analyses showed an association between the American Society of Anaesthesiologists (ASA) physical status classification III with pain (p = 0.03) and ASA III with time to flatus (p = 0.04). CONCLUSIONS: This meta-analysis and meta-regression demonstrated that NOSES had better postoperative outcomes compared to CLS. More comprehensive reviews should be conducted on the long-term outcomes specific to the extraction site to better inform clinical practice.

Pseudogout - a rare manifestation of hungry bone syndrome after focused parathyroidectomy.

Pseudogout, also known as calcium pyrophosphate deposition disease, is a rheumatological condition arising from accumulation of calcium pyrophosphate dihydrate crystals in connective tissues. We present a case of a 56-year-old Bangladeshi woman who underwent focused right inferior parathyroidectomy for primary hyperparathyroidism from a right inferior parathyroid adenoma. On the first post-operative day, she complained of left elbow painful swelling with redness and warmth. Arthrocentesis of left elbow was done due to suspicion of septic arthritis. Two weeks prior to this surgery, she had sudden bilateral knee swelling was diagnosed in her home country of bilateral knee osteoarthritis with effusion and arthrocentesis showed no crystals. Aspiration of left elbow showed calcium pyrophosphate crystals, associated with post parathyroidectomy hypocalcemia, hypomagnesemia confirming pseudogout. Her uric acid level was normal. Bilateral wrist x-rays showed triangular fibrocartilage complex chondrocalcinosis. The patient's condition improved with colchicine and naproxen, as well as calcium and magnesium replacement. Her left elbow swelling and pain resolved. Pseudogout flare is a rare but known sequelae after parathyroidectomy. Early recognition and expeditious treatment is essential.

Deep brain stimulation therapy for Parkinson's disease using frameless stereotaxy: comparison with frame-based surgery.

BACKGROUND AND PURPOSE: Deep brain stimulation (DBS) surgery has been performed using frame-based stereotaxy traditionally; however, in recent years, it has also been performed using frameless stereotaxy. The purpose of this study was to compare the experience at our centre in performing DBS surgery using frameless surgery for patients with Parkinson's disease with that of using frame-based surgery. METHODS: Twenty-four patients with advanced Parkinson's disease underwent DBS surgery, 12 with frameless and 12 with frame-based stereotaxy. After identifying the subthalamus by microelectrode recording (MER), the DBS electrodes were implanted and connected to an implanted programmable generator in all patients. Programming was started 1 month after the operation and the outcome of the patients was followed up regularly for at least 12 months. RESULTS: After 1 year of follow-up, the patients who received frameless surgery showed no difference in the degree of improvement in clinical motor function compared with the patients who received frame-based surgery (P = 0.819); the average improvement was 60.9% and 56.9%, respectively, in the stimulation alone/medication-off state, as evaluated by the Unified Parkinson's Disease Rating Scale-III motor subscore. However, the frameless group had significantly shorter total MER time (P = 0.0127) and a smaller number of trajectories (P = 0.0096) than the frame-based group. CONCLUSIONS: Our data indicate that frameless DBS surgery has a similar outcome when compared with frame-based surgery; however, frameless surgery can decrease the operation time, MER time, and MER trajectory number.

Intrapallidal injection of 6-hydroxydopamine induced changes in dopamine innervation and neuronal activity of globus pallidus.

The globus pallidus (GP) plays an important role in basal ganglia circuitry. In contrast to the well-characterized actions of dopamine on striatal neurons, the functional role of the dopamine innervation of GP is still not clearly determined. The present study aimed to investigate the effects of intrapallidal injection of 6-hydroxydopamine (6-OHDA) on rotational behavior induced by apomorphine, on the loss of dopamine cell bodies in the substantia nigra pars compacta (SNc) and fibers in the GP and striatum and on in vivo extracellularly-recorded GP neurons in the rat. Injection of 6-OHDA into GP induced severe loss of tyrosine hydroxylase-immunoreactive (TH-IR) fibers in GP (-85%) with a reduction in the number of TH-IR cell bodies in the SNc (-52%) and fibers in the striatum (-50%). S.c. injection of apomorphine in these rats induced a moderate number of turns (26+/-6 turns/5 min). Electrophysiological recordings show that 6-OHDA injection in GP induced a significant decrease of the firing rate of GP neurons (16.02+/-1.11 versus 24.14+/-1.58 spikes/sec in control animals and 22.83+/-1.28 in sham animals, one-way ANOVA, P<0.0001) without any change in the firing pattern (chi(2)=1.03, df=4, P=0.90). Our results support the premise of the existence of collaterals of SNc dopaminergic axons projecting to the striatum and GP and that dopamine plays a role in the modulation of the firing rate but not the firing pattern of GP neurons. Our data provide important insights into the functional role of the SNc-GP dopaminergic pathway suggesting that dopamine depletion in GP may participate in the development of motor disabilities.

Prediction of fatal intracranial hemorrhage in patients with acute myeloid leukemia.

BACKGROUND: Intracranial hemorrhage (ICH) is the second leading cause of mortality in patients with acute myeloid leukemia (AML). However, the prognostic factors for ICH in AML patients are still under investigation. PATIENTS AND METHODS: A total of 841 AML patients admitted to the Department of Internal Medicine from January 1995 to December 2007 were enrolled in this study. RESULTS: There were 51 patients with ICH, median age of 51 (range 17-86), including 12 patients diagnosed as acute promyelocytic leukemia. Forty-three patients were refractory/relapsed status. ICH was localized in the supratentorium (44 cases), basal ganglion (9), cerebellum (5), and brainstem (4). Twenty-one patients had multiple sites. Thirty-eight patients had intraparenchymal hemorrhage, 16 subarachnoid hemorrhage (SAH), 10 subdural hemorrhage, and one epidural hemorrhage (EDH). Hemorrhage ruptured into the ventricles in 13 patients. Thirty-four patients (67%) died of ICH within 30 days of diagnosis. Multivariate analysis revealed four independent prognostic factors, prolonged prothrombin time international normalized ratio >1.5 (P < 0.001), brainstem hemorrhage (P = 0.001), SAH (P = 0.017), and EDH (P = 0.014). Other clinico-laboratory data had no impact on 30-day survival. CONCLUSIONS: ICH has high morbidity and mortality in AML. Early detection and aggressive correction coagulopathy may prevent the catastrophic event. Prompt image study for locations and types of ICH can predict outcomes.

Expression of aminopeptidase N in bile canaliculi: a predictor of clinical outcome in biliary atresia and a potential tool to implicate the mechanism of biliary atresia.

BACKGROUND: Only a few studies on extrahepatic biliary atresia (BA) have reported that the morphological changes of bile canaliculi could predict the clinical outcome after portoenterostomy and provide differential diagnosis of neonatal jaundice. Aminopeptidase N (APN) is an ectoenzyme of bile canaliculi that is involved in bile secretion. In this study, we tried to see whether APN of bile canaliculi had a significant role in BA. PATIENTS AND METHODS: We used monoclonal antibody 9B2 to compare the expression of APN in livers with BA, neonatal hepatitis, and choledochal cysts, as well as in nontumorous portions of pediatric hepatic livers with tumors. The expression of APN in fetuses, preterm babies, and term neonates was also studied. RESULTS: A high degree of 9B2 expression in BA was closely related to poor outcome. Cholestasis in choledochal cysts, rather than neonatal hepatitis, made 9B2 expression stronger. Increasing expression of 9B2 from fetuses to neonates was noted and the degree of 9B2 expression was similar between term neonates and nontumorous portions of pediatric livers with tumors. Interestingly, some cases of BA had 9B2 expression like that of preterm babies. CONCLUSIONS: APN of bile canaliculi progressively develops from fetuses to neonates and is well developed in neonates. APN can be induced to stronger expression by obstructive jaundice. The amount of expression of APN of bile canaliculi in BA is a predictor of clinical outcome and may be a tool for implicating the mechanism of BA.

Characterization of the allosteric anion-binding site of O-acetylserine sulfhydrylase.

A new crystal structure of the A-isozyme of O-acetylserine sulfhydrylase-A (OASS) with chloride bound to an allosteric site located at the dimer interface has recently been determined [Burkhard, P., Tai, C.-H., Jansonius, J. N., and Cook, P. F. (2000) J. Mol. Biol. 303, 279-286]. Data have been obtained from steady state and presteady-state kinetic studies and from UV-visible spectral studies to characterize the allosteric anion-binding site. Data obtained with chloride and sulfate as inhibitors indicate the following: (i) chloride and sulfate prevent the formation of the external aldimines with L-cysteine or L-serine; (ii) chloride and sulfate increase the external aldimine dissociation constants for O-acetyl-L-serine, L-methionine, and 5-oxo-L-norleucine; (iii) chloride and sulfate bind to the allosteric site in the internal aldimine and alpha-aminoacrylate external aldimine forms of OASS; (iv) sulfate also binds to the active site. Sulfide behaves in a manner identical to chloride and sulfate in preventing the formation of the L-serine external aldimine. The binding of chloride to the allosteric site is pH independent over the pH range 7-9, suggesting no ionizable enzyme side chains ionize over this pH range. Inhibition by sulfide is potent (K(d) is 25 microM at pH 8) suggesting that SH(-) is the physiologic inhibitory species.

Pyridoxal 5'-phosphate-dependent alpha,beta-elimination reactions: mechanism of O-acetylserine sulfhydrylase.

O-Acetylserine sulfhydrylase catalyzes the replacement of the beta-acetoxy group of O-acetyl-L-serine with sulfide to generate L-cysteine. The reaction represents the final step in the biosynthesis of L-cysteine in enteric bacteria and plants. A quinonoid intermediate has not been detected using a variety of kinetic and spectroscopic probes for the wild-type or mutant enzymes, ruling out an E1 mechanism. The structure of the external Schiff base intermediate indicates an anti elimination. O-Acetylserine sulfhydrylase is the only known pyridoxal 5'-phosphate-dependent enzyme that catalyzes a beta-elimination reaction to have an anti E2 mechanism.

Electroacupuncture-induced neural activation detected by use of manganese-enhanced functional magnetic resonance imaging in rabbits.

OBJECTIVE: To investigate the effects of acupuncture on neural activity detected by use of manganese-enhanced functional magnetic resonance imaging (fMRI) and elucidate the relationship between somatic acupoint stimulation and brain activation. ANIMALS: 40 New Zealand White rabbits. PROCEDURE: Manganese-enhanced fMRI was performed in anesthetized rabbits manipulated with electroacupuncture (EA) on Zusanli (ST-36) and Yanglingquan (GB-34) acupoints. Image acquisition was performed on a 1.5T superconductive clinical scanner with a circular polarized extremity coil. T1-weighted images were acquired sequentially as follows: baseline, after mannitol injection, after manganese infusion, and 5 and 20 minutes after initiation of EA. RESULTS: Changes in focal neural activity were detected by use of manganese-enhanced fMRI. Stimulation on Zusanli (ST-36) for 5 minutes resulted in activation of the hippocampus, whereas stimulation on Yanglingquan (GB-34) resulted in activation of the hypothalamus, insula, and motor cortex. Activation became less specific after 20 minutes of EA. Furthermore, stimulation on ipsilateral acupoints led to bilateral brain activation. CONCLUSIONS AND CLINICAL RELEVANCE: Each acupoint has a corresponding cerebral linkage, and stimulation on these points resulted in time-dependent neural activation. Understanding the linkage between peripheral acupoint stimulation and central neural pathways may provide a useful guide for clinical applications of acupuncture.

Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound.

A new crystal structure of O-acetylserine sulfhydrylase (OASS) has been solved with chloride bound at an allosteric site and sulfate bound at the active site. The bound anions result in a new "inhibited" conformation, that differs from the "open" native or "closed" external aldimine conformations. The allosteric site is located at the OASS dimer interface. The new inhibited structure involves a change in the position of the "moveable domain" (residues 87-131) to a location that differs from that in the open or closed forms. Formation of the external aldimine with substrate is stabilized by interaction of the alpha-carboxyl group of the substrate with a substrate-binding loop that is part of the moveable domain. The inhibited conformation prevents the substrate-binding loop from interacting with the alpha-carboxyl group, and hinders formation of the external Schiff base and thus subsequent chemistry. Chloride may be an analog of sulfide, the physiological inhibitor. Finally, these results suggest that OASS represents a new class of PLP-dependent enzymes that is regulated by small anions.

Monitoring of the levodopa concentration-response relationship in Parkinson's disease.

Motor fluctuations and abnormal involuntary movements are common complications encountered in advanced Parkinson's disease (PD) patients with long-term levodopa therapy. Monitoring of plasma levodopa concentrations and clinical effects has been reported to benefit the management of these complications. However, to our knowledge, there is no data available in Taiwan concerning the correlation between the plasma levodopa levels and motor fluctuations. In this study, we developed the laboratory methodology for plasma levodopa determination by using the aluminum extraction procedure and HPLC-ED. Serial blood samples and motor scores were obtained from 7 PD patients, and the correlation between plasma levodopa levels and motor responses were studied individually. In three patients with wearing-off phenomenon, plasma levodopa concentrations are compatible with the clinical "on" and "off" states. In the other four patients with complex fluctuating responses, their levodopa dosages were adjusted by the results of monitoring. Better motor responses were achieved by optimization of the levodopa pharmacokinetics in these patients. Our preliminary data suggest that simultaneous monitoring of plasma levodopa concentrations and clinical effects might be helpful to improve the therapeutic strategy in some of the parkinsonian patients with fluctuating responses to levodopa.

O-acetylserine sulfhydrylase.

The 31P NMR data suggest slight differences in the structures around the 5'-P for the internal Schiff base and the lanthionine external Schiff base (both largely ketoeneamine) and a large difference for enolimine portion of the serine external Schiff base. Addition of cysteine or serine increase delayed fluorescence and triplet to singlet energy transfer. Addition of OAS exhibits a splitting of the 0,0 vibronic, the result of two distinct conformations, likely enolimine and ketoeneamine tautomers. Nonetheless, the alpha-amino-acrylate Schiff base conformation differs from either the internal or external Schiff base conformations. All of the time-resolved fluorescence data are consistent with conformation changes reflecting redistribution of ketoeneamine and enolimine tautomers as catalysis occurs. It is important to remember that the structural changes are substantial. The native structure (internal Schiff base) is active site open, while the K41A mutant enzyme (ketoeneamine external Schiff base) is active site closed. The trigger for the conformational change from open to closed as one goes from the internal to external Schiff base is the occupancy of the alpha-carboxyl subsite of the active site (Burkhard et al., 1999). Associated with this, as observed in pH-rate profiles, pH-dependent changes in phosphorescence, and pH-dependent changes in fluorescence enhancement upon binding acetate or cysteine is an enzyme group with a pK in the range 7-8. Dependent on the protonation state of the enzyme group, structural changes likely occur that also reflect a redistribution of the tautomeric equilibrium. Finally, the minimal catalytic cycle can likely be pictured as shown in Fig. 20. The changes may be pH dependent, and the open conformations for the internal Schiff base and the alpha-aminoacrylate Schiff base are not identical structurally, as expected because of the increased stability of the latter.

pH-induced intramolecular electron transfer between the iron-sulfur protein and cytochrome c(1) in bovine cytochrome bc(1) complex.

Structural analysis of the bc(1) complex suggests that the extra membrane domain of iron-sulfur protein (ISP) undergoes substantial movement during the catalytic cycle. Binding of Qo site inhibitors to this complex affects the mobility of ISP. Taking advantage of the difference in the pH dependence of the redox midpoint potentials of cytochrome c(1) and ISP, we have measured electron transfer between the [2Fe-2S] cluster and heme c(1) in native and inhibitor-treated partially reduced cytochrome bc(1) complexes. The rate of the pH-induced cytochrome c(1) reduction can be estimated by conventional stopped-flow techniques (t1/2, 1-2 ms), whereas the rate of cytochrome c(1) oxidation is too high for stopped-flow measurement. These results suggest that oxidized ISP has a higher mobility than reduced ISP and that the movement of reduced ISP may require an energy input from another component. In the 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)-inhibited complex, the rate of cytochrome c(1) reduction is greatly decreased to a t1/2 of approximately 2.8 s. An even lower rate is observed with the stigmatellin-treated complex. These results support the idea that UHDBT and stigmatellin arrest the [2Fe-2S] cluster at a fixed position, 31 A from heme c(1), making electron transfer very slow.

Substitution of pyridoxal 5'-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis.

D-Serine dehydratase (DSD) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of D-serine to pyruvate and ammonia. Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues. PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive. The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm. The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base. The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm. In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species. The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visible absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity. The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue).

Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium.

Covalent binding of L-methionine as an external aldimine to the pyridoxal 5'-phosphate-cofactor in the K41A mutant of O-acetylserine sulfhydrylase from Salmonella typhimurium induces a large conformational change in the protein. Methionine mimics the action of the substrate O-acetyl-L-serine during catalysis. The alpha-carboxylate moiety of L-methionine in external aldimine linkage with the active site pyridoxal 5'-phosphate forms a hydrogen bonding network to the "asparagine-loop" P67-T68-N69-G70 which adopts a different conformation than in the native protein. The side-chain nitrogen of Asn69 moves more than 7 A to make a hydrogen bond to the alpha-carboxylate group of the inhibitor. As the external aldimine is formed, the PLP tilts by 13 degrees along its longitudinal axis such that C4' moves toward the entrance to the active site and the side-chain of the methionine is directed toward the active site entrance. The local rearrangement acts as a trigger to induce a large global conformational change in the protein. A subdomain comprised of beta-strand 4, alpha-helix 3, beta-strand 5 and alpha-helix 4 moves towards the active site by a rotation of 7 degrees. This subdomain movement results in a reduction of the severe twist of its central beta-sheet and reduces the active site entrance to a small hole, giving access only to small molecules like sulfide, the second substrate, or acetate, the first product.

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.

Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate.

O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5'-phosphate (PLP) dependent enzyme from Salmonella typhimurium that catalyzes the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide to give L-cysteine. The reaction occurs via a ping-pong kinetic mechanism in which alpha-aminoacrylate in Schiff base with the active site PLP is an intermediate [Cook, P. F., Hara, S., Nalabolu, S. R., and Schnackerz, K. D. (1992) Biochemistry 31, 2298-2303]. The sequence around the Schiff base lysine (K41) has been determined [Rege, V. D., Kredich, N. M., Tai, C.-H., Karsten, W. E., Schnackerz, K. D., & Cook, P. F. (1996) Biochemistry 35, 13485-13493], and the sole cysteine in the primary structure is immediately C-terminal to the lysine. In an effort to assess the role of C42, it has been changed to serine and alanine by site-directed mutagenesis. The mutant proteins are structurally nearly identical to the wild-type enzyme on the basis of UV-visible, fluorescence, far-UV and cofactor-induced CD, and 31P NMR studies, but subtle structural differences are noted. Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme. The C42S mutant exhibits a > 50-fold increase in the OAS:acetate lyase activity and a 17-fold decrease in V for the cysteine synthesis compared to the wild-type enzyme, while decreases of > 200-fold in the OAS: acetate lyase activity and a 30-fold decrease in V for the cysteine synthesis are found for the C42A mutant enzyme. In both cases, however, the pH dependence of kinetic parameters for cysteine synthesis and OAS: acetate lyase activity yield, within error, identical pK values. In the three-dimensional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protein. The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized movement of the serine hydroxyl (compared to the cysteine thiol) toward additional hydrophilic, hydrogen-bonding groups in C42S, or away from hydrophilic groups for C42A, repositioning structure around and including K41. Subtle movement of the epsilon-amino group of K41 may change the geometry for nucleophilic displacement of the amino acid from PLP, leading to changes in overall activity and stability of the alpha-aminoacrylate intermediate. Data indicate that single amino acid substitutions that yield only subtle changes in structure can produce large differences in reaction rates and overall mechanism.

Substitution of pyridoxal 5'-phosphate in the O-acetylserine sulfhydrylase from Salmonella typhimurium by cofactor analogs provides a test of the mechanism proposed for formation of the alpha-aminoacrylate intermediate.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step in the de novo synthesis of L-cysteine in Salmonella typhimurium. Complementary cofactor mutagenesis in which the active site PLP is substituted with cofactor analogs is used to test the mechanism proposed for the OASS. Data obtained with the pyridoxal 5'-deoxymethylenephosphonate-substituted enzyme suggest that the binding of OAS as it forms the external Schiff base is such that the acetate side chain is properly positioned for elimination (orthogonal to the developing alpha,beta-double bond) only about 1% of the time. Data support the assignment of an enzyme group with a pK of 6.7 that interacts with the acetyl side chain, maintaining it orthogonal to the developing alpha,beta-double bond. Similar studies of the 2'-methylpyridoxal 5'-phosphate-substituted enzyme suggest that, although the mechanism is identical to that catalyzed by native OASS, the reaction coordinate for alpha-proton abstraction may be decreased compared with that observed for the native enzyme.