Understanding Protein Lids: Kinetic Analysis of Active Hinge Mutants in Triosephosphate Isomerase^†

In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495−1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that kcat/Km's are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed

Alternating Ring-Opening Metathesis Polymerization (AROMP) of Hydrophobic and Hydrophilic Monomers Provides Oligomers with Side-Chain Sequence Control

We report the formation of oligomers with side-chain sequence control using ruthenium-catalyzed alternating ring-opening metathesis polymerization (AROMP). These oligomers are prepared through sequential, stoichiometric addition of bicyclo[4.2.0]­oct-1(8)-ene-8-carboxamide (monomer A) at 85 °C and cyclohexene (monomer B) at 45 °C to generate sequences up to 24 monomeric units composed of (A-alt-B)n and (A′-alt-B)n microblocks, where n ranges from 1 to 6. Herein, monomer A has an alkyl side chain, and monomer A′ has a glycine methyl ester side chain. Increasing microblock size from one to six results in an increasing water contact angle on spin-coated thin films, despite the constant ratio of hydrophilic and hydrophobic moieties. However, a disproportionately high contact angle was observed when n equals 2. Thus, the unique all-carbon backbone formed in the AROMP of bicyclo[4.2.0]­oct-1(8)-ene-8-carboxamides and cyclohexene provides a platform for the nontemplated preparation of materials with specific sequences of side chains

Substituent Effects Provide Access to Tetrasubstituted Ring-Opening Olefin Metathesis of Bicyclo[4.2.0]oct-6-enes

Herein, we report the origin of unexpected reactivity of bicyclo[4.2.0]­oct-6-ene substrates containing an α,β-unsaturated amide moiety in ruthenium-catalyzed alternating ring-opening metathesis polymerization reactions. Specifically, compared with control substrates bearing an ester, alkyl ketone, nitrile, or tertiary amide substituent, α,β-unsaturated substrates with a weakly acidic proton showed increased rates of ring-opening metathesis mediated by Grubbs-type ruthenium catalysts. 1H NMR and IR spectral analyses indicated that deprotonation of the α,β-unsaturated amide substrates resulted in stronger coordination of the carbonyl group to the ruthenium metal center. Principal component analysis identified ring strain and the electron density on the carbonyl oxygen (based on structures optimized by means of ωB97X-D/6311+G­(2df,2p) calculations) as the two key contributors to fast ring-opening metathesis of the bicyclo[4.2.0]­oct-6-enes; whereas the dipole moment, conjugation, and energy of the highest occupied molecular orbital had little to no effect on the reaction rate. We conclude that alternating ring-opening metathesis polymerization reactions of bicyclo[4.2.0]­oct-6-enes with unstrained cycloalkenes require an ionizable proton for efficient generation of alternating polymers

Increased Polymer Length of Oligopeptide-Substituted Polynorbornenes with LiCl

The ring-opening metathesis polymerization (ROMP) reaction is extraordinarily useful for the preparation of a large variety of polymers. We report that the length (n = 25−50) of high-substituent-density oligopeptide polymers synthesized by ROMP is dramatically improved upon addition of LiCl to reduce polymer and oligopeptide aggregation. This methodology should significantly expand the variety of polymers that may be prepared by ROMP and be of general use with norbornyl oligopeptides of any sequence

Increased Polymer Length of Oligopeptide-Substituted Polynorbornenes with LiCl

The ring-opening metathesis polymerization (ROMP) reaction is extraordinarily useful for the preparation of a large variety of polymers. We report that the length (n = 25−50) of high-substituent-density oligopeptide polymers synthesized by ROMP is dramatically improved upon addition of LiCl to reduce polymer and oligopeptide aggregation. This methodology should significantly expand the variety of polymers that may be prepared by ROMP and be of general use with norbornyl oligopeptides of any sequence

A Facile Synthetic Method to Prepare Fluorescently Labeled ROMP Polymers

To probe the activities of sperm ADAM protein (fertilinβ), we devised a general synthetic strategy to generate fluorescently labeled fertilinβ oligopeptide polymers. Immunofluorescence studies with these polymers demonstrated that fertilinβ polymers bind specifically to a protein receptor on the mouse egg plasma membrane

A C₆-Flavin Adduct Is the Major Product of Irreversible Inactivation of Cholesterol Oxidase by 2α,3α-Cyclopropano-5α-cholestan-3β-ol

We have synthesized 2α,3α-cyclopropano-5α-cholestan-3β-ol and tested it as a substrate and inhibitor of cholesterol oxidase. The cyclopropylsterol irreversibly inhibits cholesterol oxidase with a kinact = 0.010 min-1 and Ki = 36 μM. Efficient inactivation requires the general base His447. Two FAD−steroid adducts were isolated by reversed-phase HPLC. The UV/vis, fluorescence and mass spectra of the adducts suggest that the FAD cofactor acts as an electrophile in a cyclopropoxide ring-opening reaction to form a C6-alkylated flavin (68%) and either an N5 flavin adduct or a cyclic N5−C4a flavin adduct (32%). Cyclopropoxide ring-opening to form a C6−FAD adduct represents a new approach to flavoenzyme inhibition

4,5-Cyclopropanocholestan-3β-ol Substrates for Cholesterol Oxidase and Their ¹H NMR Assignments

We have assayed 4,5-cyclopropanocholestan-3-ols and 4,5-cyclopropanocholestan-3-ones and tested them as substrates and inhibitors of cholesterol oxidase. The 4,5-cyclopropanocholestan-3β-ols (α and β) are substrates of cholesterol oxidase that are converted to their respective ketones 1000-fold more slowly than cholesterol. The induced ring-current effects of a cyclopropane ring are clearly illustrated in the 1H NMR spectra of these sterols. These shielding effects are dramatic because of the rigidity of the steroid backbone. Assignments of the 1H resonances of the A, B, and cyclopropyl rings of the sterols have been made using DQF-COSY and NOESY experiments. We have assigned the upfield multiplet at approximately 0.5 ppm to H6α in both isomers. H6α is shielded by the cyclopropyl σ bond. H6β is deshielded by the cyclopropane ring and appears at approximately 2.0 ppm in both isomers

A GMC Oxidoreductase Homologue Is Required for Acetylation of Glycopeptidolipid in <i>Mycobacterium smegmatis</i>

The <i>Mycobacterium tuberculosis Rv3409c</i> gene is required for modulation of the Toll-like receptor 2 (TLR-2) signaling response in infected macrophages. Although each is annotated as encoding a cholesterol oxidase, neither <i>Rv3409c</i> nor its ortholog <i>MSMEG1604</i> is required for the metabolism of cholesterol in mycobacteria. Here we report that a unique lipid, L1334, accumulates in a MSMEG1604 transposon mutant in the <i>Mycobacterium smegmatis</i> cell envelope. L1334 is a polar glycopeptidolipid that is hyperrhamnosylated and in which the 6-deoxytalose moiety is not acetylated. The alteration of L1334 acetylation is consistent with a reduced level of interference with TLR-2 signaling in mutant infected macrophages

Fucose, Mannose, and β‑<i>N</i>‑Acetylglucosamine Glycopolymers Initiate the Mouse Sperm Acrosome Reaction through Convergent Signaling Pathways

The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-<i>N</i>-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca²⁺-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR