Lysine164α of protein farnesyltransferase is important for both CaaX substrate binding and catalysis

2001 ◽  
Vol 360 (3) ◽  
pp. 625-631 ◽  
Author(s):  
Kendra E. HIGHTOWER ◽  
Smita DE ◽  
Carolyn WEINBAUM ◽  
Rebecca A. SPENCE ◽  
Patrick J. CASEY
Keyword(s):  
Rate Constant ◽  
Positive Charge ◽  
Substrate Binding ◽  
Product Formation ◽  
Chemical Mechanism ◽  
Α Subunit ◽  
Protein Farnesyltransferase ◽  
Oncogenic Ras ◽  
Tumour Formation ◽  
Mechanism Of Catalysis

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys164 of the α subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys164α is involved in formation of the enzyme (E)·farnesyl diphosphate (FPP)·peptide ternary complex. Furthermore, mutation of Lys164α resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys164α, as well as the positioning of the charge, are important for overall catalysis by FTase.

Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽  
2021 ◽  
Author(s):  
Margrethe Gaardløs ◽  
Sergey A Samsonov ◽  
Marit Sletmoen ◽  
Maya Hjørnevik ◽  
Gerd Inger Sætrom ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Interdisciplinary Approach ◽  
Product Formation ◽  
Titration Calorimetry ◽  
Binding Groove ◽  
Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

2001 ◽  
Vol 130-132 ◽  
pp. 15-28 ◽  
Author(s):  
Stéphane Marchal ◽  
David Cobessi ◽  
Sophie Rahuel-Clermont ◽  
Frédérique Tête-Favier ◽  
André Aubry ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Aldehyde Dehydrogenase ◽  
Binding Sites ◽  
Substrate Binding ◽  
Chemical Mechanism ◽  
Substrate Binding Sites

scholarly journals Lysine164α of protein farnesyltransferase is important for both CaaX substrate binding and catalysis

2001 ◽  
Vol 360 (3) ◽  
pp. 625 ◽  
Author(s):  
Kendra E. HIGHTOWER ◽  
Smita DE ◽  
Carolyn WEINBAUM ◽  
Rebecca A. SPENCE ◽  
Patrick J. CASEY
Keyword(s):  
Substrate Binding ◽  
Protein Farnesyltransferase

scholarly journals Substrate Binding Is Required for Release of Product from Mammalian Protein Farnesyltransferase

1997 ◽  
Vol 272 (15) ◽  
pp. 9989-9993 ◽  
Author(s):  
William R. Tschantz ◽  
Eric S. Furfine ◽  
Patrick J. Casey
Keyword(s):  
Substrate Binding ◽  
Protein Farnesyltransferase ◽  
Mammalian Protein

Presteady-state kinetics of Bacillus 1,3–1,4-β-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate

2001 ◽  
Vol 357 (1) ◽  
pp. 195-202
Author(s):  
Mireia ABEL ◽  
Antoni PLANAS ◽  
Ulla CHRISTENSEN
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Product Formation ◽  
Lag Phase ◽  
Wild Type ◽  
Induced Fit ◽  
Enzyme Substrate ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Presteady State Kinetics

In the present study the first stopped-flow experiments performed on Bacillus 1,3–1,4-β-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-β-cellobiosyl-β-d-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100s−1, was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme–substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

scholarly journals Solvation dynamics-powered structure and function of multi-molecular cellular systems exemplified by non-equilibrium cereblon-degrader-CK1α ternary complex formation

2021 ◽  
Author(s):  
Hongbin Wan ◽  
Vibhas Aravamuthan ◽  
Sarah Williams ◽  
Charles Wartchow ◽  
Jose Duca ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Rate Constant ◽  
Surface Composition ◽  
Substrate Binding ◽  
Structure And Function ◽  
Enzyme Activation ◽  
Cellular Systems ◽  
State Transitions ◽  
Solvation Dynamics ◽  
And Function

Cellular functions are executed via a form of analog computing that is based on the switchable covalent and non-covalent states of multi-molecular fluxes (i.e., time-dependent species/state concentrations) operating in the non-linear dynamics regime. We and others have proposed that the non-covalent states and state transitions of aqueous fluxes are powered principally by the storage and release of potential energy to/from the anisotropic H-bond network of solvating water (which we refer to as the 'solvation field'), which is a key tenet of a first principles theory on cellular structure and function (called Biodynamics) that we outlined previously. This energy is reflected in water occupancy as a function of solute surface position, which can be probed computationally using WATMD software. In our previous work, we used this approach to deduce the structural dynamics of the COVID main protease, including substrate binding-induced enzyme activation and dimerization, and product release-induced dimer dissociation. Here, we examine: 1) The general relationships between surface composition/topology and solvation field properties for both high and low molecular weight (HMW and LMW) solutes. 2) The general means by which structural dynamics are powered by solvation free energy, which we exemplify via binding between the E3 ligase CUL4A/RBX1/DDB1/CRBN, LMW degraders, and substrates. We propose that degraders organize the substrate binding surface of cereblon toward complementarity with native and neo substrates, thereby speeding the association rate constant and incrementally slowing the dissociation rate constant. 3) Structure-activity relationships (SAR) based on complementarity between the solvation fields of cognate protein-ligand partners exemplified via LMW degraders.

Yeast protein farnesyltransferase: steady-state kinetic studies of substrate binding

Biochemistry ◽  
1995 ◽  
Vol 34 (51) ◽  
pp. 16687-16694 ◽  
Author(s):  
Julia M. Dolence ◽  
Pamela B. Cassidy ◽  
Jeffery R. Mathis ◽  
C. Dale Poulter
Keyword(s):  
Steady State ◽  
Substrate Binding ◽  
Kinetic Studies ◽  
Yeast Protein ◽  
Protein Farnesyltransferase ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals Errors in the evaluation of Arrhenius and van't Hoff plots

1983 ◽  
Vol 209 (1) ◽  
pp. 277-280 ◽  
Author(s):  
T Keleti
Keyword(s):  
Rate Constant ◽  
Arrhenius Plot ◽  
Product Formation ◽  
Hoff Plot

Errors in the numerical values of activation or normal enthalpies, entropies and free enthalpies calculated from Arrhenius or van't Hoff plots, respectively, are due to the neglect of equidimensionality in equations, or to inappropriate approximations. The logarithmization of dimensioned quantities should be avoided, which demands the use of relative concentrations if a change in mole number occurs in the reaction. The application of the Arrhenius plot to enzymic reactions by using Vmax./ET instead of the rate constant of product formation has meaning only if the reaction follows the simplest Michaelis-Menten mechanism; however, the use of the van't Hoff plot using Km is questionable even in the latter case.

scholarly journals A Single Active-Site Mutation of P450BM-3 Dramatically Enhances Substrate Binding and Rate of Product Formation

Biochemistry ◽  
2011 ◽  
Vol 50 (39) ◽  
pp. 8333-8341 ◽  
Author(s):  
Donovan C. Haines ◽  
Amita Hegde ◽  
Baozhi Chen ◽  
Weiqiang Zhao ◽  
Muralidhar Bondlela ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Product Formation ◽  
Site Mutation
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