23 research outputs found
Glycosyl Cations versus Allylic Cations in Spontaneous and Enzymatic Hydrolysis
Enzymatic
prenyl and glycosyl transfer are seemingly unrelated
reactions that yield molecules and protein modifications with disparate
biological functions. However, both reactions employ diphosphate-activated
donors and each proceed via cationic species: allylic cations and
oxocarbenium ions, respectively. In this study, we explore the relationship
between these processes by preparing valienyl ethers to serve as glycoside
mimics that are capable of allylic rather than oxocarbenium cation
stabilization. Rate constants for spontaneous hydrolysis of aryl glycosides
and their analogous valienyl ethers were found to be almost identical,
as were the corresponding activation enthalpies and entropies. This
close similarity extended to the associated secondary kinetic isotope
effects (KIEs), indicating very similar transition state stabilities
and structures. Screening a library of over 100 β-glucosidases
identified a number of enzymes that catalyze hydrolysis of these valienyl
ethers with <i>k</i><sub>cat</sub> values up to 20 s<sup>â1</sup>. Detailed analysis of one such enzyme showed that
ether hydrolysis occurs via the analogous mechanisms found for glycosides,
and through a very similar transition state. This suggests that the
generally lower rates of enzymatic cleavage of the cyclitol ethers
reflects evolutionary specialization of these enzymes toward glycosides
rather than inherent reactivity differences
Glycoside Cleavage by a New Mechanism in Unsaturated Glucuronyl Hydrolases
Unsaturated glucuronyl hydrolases (UGLs) from GH family 88 of the CAZy classification system cleave a terminal unsaturated sugar from the oligosaccharide products released by extracellular bacterial polysaccharide lyases. This pathway, which is involved in extracellular bacterial infection, has no equivalent in mammals. A novel mechanism for UGL has previously been proposed in which the enzyme catalyzes hydration of a vinyl ether group in the substrate, with subsequent rearrangements resulting in glycosidic bond cleavage. However, clear evidence for this mechanism has been lacking. In this study, analysis of the products of UGL-catalyzed reactions in water, deuterium oxide, and dilute methanol in water, in conjunction with the demonstration that UGL rapidly cleaves thioglycosides and glycosides of inverted anomeric configuration (substrates that are resistant to hydrolysis by classical glycosidases), provides strong support for this new mechanism. A hydration-initiated process is further supported by the observed UGL-catalyzed hydration of a C-glycoside substrate analogue. Finally, the observation of a small β-secondary kinetic isotope effect suggests a transition state with oxocarbenium ion character, in which the hydrogen at carbon 4 adopts an axial geometry. Taken together, these observations validate the novel vinyl ether hydration mechanism and are inconsistent with either inverting or retaining direct hydrolase mechanisms at carbon 1
Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic ÎąâAmylase
Starch provides the major source
of caloric intake in many diets.
Cleavage of starch into malto-oligosaccharides in the gut is catalyzed
by pancreatic Îą-amylase. These oligosaccharides are then further
cleaved by gut wall Îą-glucosidases to release glucose, which
is absorbed into the bloodstream. Potential surface binding sites
for starch on the pancreatic amylase, distinct from the active site
of the amylase, have been identified through X-ray crystallographic
analyses. The role of these sites in the degradation of both starch
granules and soluble starch was probed by the generation of a series
of surface variants modified at each site to disrupt binding. Kinetic
analysis of the binding and/or cleavage of substrates ranging from
simple maltotriosides to soluble starch and insoluble starch granules
has allowed evaluation of the potential role of each such surface
site. In this way, two key surface binding sites, on the same face
as the active site, are identified. One site, containing a pair of
aromatic residues, is responsible for attachment to starch granules,
while a second site featuring a tryptophan residue around which a
malto-oligosaccharide wraps is shown to heavily influence soluble
starch binding and hydrolysis. These studies provide insights into
the mechanisms by which enzymes tackle the degradation of largely
insoluble polymers and also present some new approaches to the interrogation
of the binding sites involved
Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening
In vitro compartmentalization-based
fluorescence-activated cell
sorting (IVC-FACS) is a powerful screening tool for directed evolution
of enzymes. However, the efficiency of IVC-FACS is limited by the
tendency of the fluorescent reporter to diffuse out of the droplets,
which decouples the genotype and phenotype of the target gene. Herein
we present a new strategy called fluorescence droplet entrapment (FDE)
to solve this problem. The substrate is designed with a polarity that
enables it to pass through the oil phase, react with the enzyme and
generate an oil-impermeable and fluorescent product that remains entrapped
inside the droplet. Several FDE substrates were designed, using two
distinct substrate engineering strategies, for the detection of phosphotriesterases,
carboxylesterases, and glycosidases activities. Model screening assays
in which rare phosphotriesterase-active cells were enriched from large
excesses of inactive cells showed that the enrichment efficiency achievable
using an FDE substrate was as high as 900-fold: the highest yet reported
in such an IVC-FACS system. Thus, FDE provides a means to tightly
control the onset of the enzymatic reaction, minimize droplet cross-talk,
and lower the background fluorescence. It therefore may serve as a
useful strategy for the IVC-FACS screening of enzymes, antibodies,
and other proteins
Remarkable Reactivity Differences between Glucosides with Identical Leaving Groups
Two isomeric aryl
2-deoxy-2-fluoro-β-glucosides react with
a β-glucosidase at rates differing by 10<sup>6</sup>-fold, despite
the fact that they release the same aromatic aglycone. In contrast,
the equivalent glucoside substrates react with essentially identical
rate constants. Insight into the source of these surprising rate differences
was obtained through a comprehensive study of the nonenzymatic (spontaneous)
hydrolysis of these same substrates, wherein an approximate 10<sup>5</sup>-fold difference in rates was measured, clarifying that the
differences were inherent rather than being due to specific interactions
with the enzyme. The possibility that an alternate nucleophilic aryl
substitution mechanism was responsible for the rapid reaction of the
faster substrate was excluded through <sup>18</sup>O-labeling studies.
Further exploration of the origins of these rate differences involved
analysis of X-ray crystal structures as well as quantum chemical calculations,
which surprisingly revealed that ground state destabilization and
transition state stabilizing effects contribute almost equally to
the observed reactivity differences. These studies highlight the dangers
of using simple reference equilibria such as p<i>K</i><sub>a</sub> values as measures of leaving group ability
Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids
Understanding the detailed mechanisms
of enzyme-catalyzed hydrolysis
of the glycosidic bond is fundamentally important, not only to the
design of tailored cost-efficient, stable and specific catalysts but
also to the development of specific glycosidase inhibitors as therapeutics.
Retaining glycosidases employ two key carboxylic acid residues, typically
glutamic acids, in a double-displacement mechanism involving a covalent
glycosyl-enzyme intermediate. One Glu functions as a nucleophile while
the other acts as a general acid/base. A significant part of enzymatic
proficiency is attributed to a âperfect matchâ of the
electrostatics provided by these key residues, a hypothesis that has
been remarkably difficult to prove in model systems or in enzymes
themselves. We experimentally probe this synergy by preparing synthetic
variants of a model glycosidase <i>Bacillus circulans</i> β-xylanase (Bcx) with the nucleophile Glu78 substituted by
4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity.
These Bcx variants were semisynthesized by preparation of optically
pure fluoroglutamic acid building blocks, incorporation into synthetic
peptides, and ligation onto a truncated circular permutant of Bcx.
By measuring the effect of altered electrostatics in the active site
on enzyme kinetic constants, we show that lowering the nucleophile
p<i>K</i>a by two units shits the pH-dependent activity
by one pH unit. Linear free energy correlations using substrates of
varying leaving group ability indicate that by reducing nucleophilic
catalysis the concerted mechanism of the enzyme is disrupted and shifted
toward a dissociative pathway. Our study represents the first example
of site-specific introduction of fluorinated glutamic acids into any
protein. Furthermore, it provides unique insights into the synergy
of nucleophilic and acid/base catalysis within an enzyme active site
Mechanistic Insights into the 1,3-Xylanases: Useful Enzymes for Manipulation of Algal Biomass
Xylanases capable of degrading the crystalline microfibrils
of
1,3-xylan that reinforce the cell walls of some red and siphonous
green algae have not been well studied, yet they could prove to be
of great utility in algaculture for the production of food and renewable
chemical feedstocks. To gain a better mechanistic understanding of
these enzymes, a suite of reagents was synthesized and evaluated as
substrates and inhibitors of an <i>endo</i>-1,3-xylanase.
With these reagents, a retaining mechanism was confirmed for the xylanase,
its catalytic nucleophile identified, and the existence of â3
to +2 substrate-binding subsites demonstrated. Protein crystal X-ray
diffraction methods provided a high resolution structure of a trapped
covalent glycosylâenzyme intermediate, indicating that the
1,3-xylanases likely utilize the <sup>1</sup><i>S</i><sub>3</sub> â <sup>4</sup><i>H</i><sub>3</sub> â <sup>4</sup><i>C</i><sub>1</sub> conformational itinerary to
effect catalysis
Rapid Assembly of a Library of Lipophilic Iminosugars via the ThiolâEne Reaction Yields Promising Pharmacological Chaperones for the Treatment of Gaucher Disease
A highly divergent route to lipophilic iminosugars that
utilizes the thiolâene reaction was developed to enable the
rapid synthesis of a collection of 16 dideoxyiminoxylitols bearing
various different lipophilic substituents. Enzyme kinetic analyses
revealed that a number of these products are potent, low-nanomolar
inhibitors of human glucocerebrosidase that stabilize the enzyme to
thermal denaturation by up to 20 K. Cell based assays conducted on
Gaucher disease patient derived fibroblasts demonstrated that administration
of the compounds can increase lysosomal glucocerebrosidase activity
levels by therapeutically relevant amounts, as much as 3.2-fold in
cells homozygous for the p.N370S mutation and 1.4-fold in cells homozygous
for the p.L444P mutation. Several compounds elicited this increase
in enzyme activity over a relatively wide dosage range. The data assembled
here illustrate how the lipophilic moiety common to many glucocerebrosidase
inhibitors might be used to optimize a lead compoundâs ability
to chaperone the protein in cellulo. The flexibility of this synthetic strategy makes it an attractive
approach to the rapid optimization of glycosidase inhibitor potency
and pharmacokinetic behavior
Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids
Understanding the detailed mechanisms
of enzyme-catalyzed hydrolysis
of the glycosidic bond is fundamentally important, not only to the
design of tailored cost-efficient, stable and specific catalysts but
also to the development of specific glycosidase inhibitors as therapeutics.
Retaining glycosidases employ two key carboxylic acid residues, typically
glutamic acids, in a double-displacement mechanism involving a covalent
glycosyl-enzyme intermediate. One Glu functions as a nucleophile while
the other acts as a general acid/base. A significant part of enzymatic
proficiency is attributed to a âperfect matchâ of the
electrostatics provided by these key residues, a hypothesis that has
been remarkably difficult to prove in model systems or in enzymes
themselves. We experimentally probe this synergy by preparing synthetic
variants of a model glycosidase <i>Bacillus circulans</i> β-xylanase (Bcx) with the nucleophile Glu78 substituted by
4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity.
These Bcx variants were semisynthesized by preparation of optically
pure fluoroglutamic acid building blocks, incorporation into synthetic
peptides, and ligation onto a truncated circular permutant of Bcx.
By measuring the effect of altered electrostatics in the active site
on enzyme kinetic constants, we show that lowering the nucleophile
p<i>K</i>a by two units shits the pH-dependent activity
by one pH unit. Linear free energy correlations using substrates of
varying leaving group ability indicate that by reducing nucleophilic
catalysis the concerted mechanism of the enzyme is disrupted and shifted
toward a dissociative pathway. Our study represents the first example
of site-specific introduction of fluorinated glutamic acids into any
protein. Furthermore, it provides unique insights into the synergy
of nucleophilic and acid/base catalysis within an enzyme active site
Rapid Assembly of a Library of Lipophilic Iminosugars via the ThiolâEne Reaction Yields Promising Pharmacological Chaperones for the Treatment of Gaucher Disease
A highly divergent route to lipophilic iminosugars that
utilizes the thiolâene reaction was developed to enable the
rapid synthesis of a collection of 16 dideoxyiminoxylitols bearing
various different lipophilic substituents. Enzyme kinetic analyses
revealed that a number of these products are potent, low-nanomolar
inhibitors of human glucocerebrosidase that stabilize the enzyme to
thermal denaturation by up to 20 K. Cell based assays conducted on
Gaucher disease patient derived fibroblasts demonstrated that administration
of the compounds can increase lysosomal glucocerebrosidase activity
levels by therapeutically relevant amounts, as much as 3.2-fold in
cells homozygous for the p.N370S mutation and 1.4-fold in cells homozygous
for the p.L444P mutation. Several compounds elicited this increase
in enzyme activity over a relatively wide dosage range. The data assembled
here illustrate how the lipophilic moiety common to many glucocerebrosidase
inhibitors might be used to optimize a lead compoundâs ability
to chaperone the protein in cellulo. The flexibility of this synthetic strategy makes it an attractive
approach to the rapid optimization of glycosidase inhibitor potency
and pharmacokinetic behavior