Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.     

Comparisons of phosphorothioate with phosphate transfer reactions for a monoester,diester, and triester: isotope effect studies

Phosphorothioate esters are sometimes used as surrogates for phosphate ester substrates in studies of enzymatic phosphoryl transfer reactions. To gain better understanding of the comparative inherent chemistry of the two types of esters, we have measured equilibrium and kinetic isotope effects for several phosphorothioate esters of p-nitrophenol (pNPPT) and compared the results with data from phosphate esters. The primary (18)O isotope effect at the phenolic group ((18)k(bridge)), the secondary nitrogen-15 isotope effect ((15)k) in the nitro group, and (for the monoester and diester) the secondary oxygen-18 isotope effect ((18)k(nonbridge)) in the phosphoryl oxygens were measured. The equilibrium isotope effect (EIE) (18)k(nonbridge) for the deprotonation of the monoanion of pNPPT is 1.015 +/- 0.002, very similar to values previously reported for phosphate monoesters. The EIEs for complexation of Zn(2+) and Cd(2+) with the dianion pNPPT(2-) were both unity. The mechanism of the aqueous hydrolysis of the monoanion and dianion of pNPPT, the diester ethyl pNPPT, and the triester dimethyl pNPPT was probed using heavy atom kinetic isotope effects. The results were compared with the data reported for analogous phosphate monoester, diester, and triester reactions. The results suggest that leaving group bond fission in the transition state of reactions of the monoester pNPPT is more advanced than for its phosphate counterpart pNPP, while alkaline hydrolysis of the phosphorothioate diester and triester exhibits somewhat less advanced bond fission than that of their phosphate ester counterparts.     

Evaluation of polyblends of cottonseed protein and polycaprolactone plasticized by cottonseed oil

Recently, there has been an increasing trend toward replacing conventional fossil-based plastics with bioplastics that are eco-friendly and biodegradable. In this work, blends of polycaprolactone (PCL) and cottonseed protein plasticized with cottonseed oil were made and analyzed for their mechanical, adhesive, and thermal properties. The addition of water-washed cottonseed meal (WCSM) to PCL increased the Young’s modulus but decreased the tensile strength and elongation-at-break of PCL. The addition of cottonseed oil to the PCL/WCSM blend kept the tensile strength about the same but enhanced the elongation. The PCL blends with WCSM and cottonseed protein isolate gave about the same mechanical properties, both somewhat better than the PCL/soy protein isolate blend. As plasticizers, cottonseed oil performed slightly better than coconut oil, both better than poly(ethylene glycol). The addition of WCSM and cottonseed oil (up to a PCL:WCSM:plasticizer ratio of 60:40:20) did not change the adhesive performance of PCL on fiberboard. Thus, the combination of PCL/cottonseed protein/cottonseed oil seems to be a viable bioplastic, and one possible application for this material may be in the hot melt adhesive area.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

Low divergence angle and low jitter 40 GHz AlGaInAs/InP 1.55 μm mode-locked lasers

We demonstrate a novel (to the best of our knowledge) 40?GHz passively mode-locked AlGaInAs/InP 1.55?μm laser with a low divergence angle (12.7°×26.3°), timing jitter of 1.2?ps (10?kHz-100?MHz), and a radio frequency linewidth of 25?kHz.     

High power (130 mW) 40 GHz 1.55 μm mode-locked distributed Bragg reflector lasers with integrated optical amplifiers

High output power 40 GHz 1.55 μm passively mode-locked surface-etched distributed Bragg reflector (DBR) lasers with monolithically integrated semiconductor optical amplifiers are reported. These are based on an optimized AlGaInAs/InP epitaxial structure with a three quantum well active layer and an optical trap layer. The device produces near transform limited Gaussian pulses with a pulse duration of 3.3 ps. An average output power during mode-locked operation of 130 mW was achieved with a corresponding peak power of >1 W.