Art is long and time is fleeting: the current problems and future prospects for time-resolved enzyme crystallography

1992 ◽  
Vol 340 (1657) ◽  
pp. 323-334 ◽  
Keyword(s):  
Enzymatic Reaction ◽  
Site Directed Mutagenesis ◽  
Kinetic Measurements ◽  
Laue Diffraction ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Time Regime ◽  
Kinetics Of ◽  
State Kinetic

I offer comments on the challenges and problems of the future based on the papers in this volume. First, the requirement of the Laue technique for a very well-ordered crystal is a major obstacle to many studies. Efforts to ease this problem are needed. Secondly, the fundamental issues in time-resolved crystallography are now chemical rather than crystallographic. Methods for the rapid initiation of many reactions must be developed. Thirdly, it is imperative that the kinetics of the process in question be studied in the crystal before any diffraction experiments are done. We need better ways to make those solid state kinetic measurements. Fourthly, we should make use of combined methods, such as cryoenzymology plus Laue diffraction or site-directed mutagenesis plus Laue diffraction, to bring many processes into the time regime in which we currently can work. Fifthly, we have to be able to deconvolute diffraction data that come from a mixture of two or three discrete species. Finally, no matter how powerful our synchrotrons get, it seems to me that some of the most important events in any enzymatic reaction are not going to be accessible: consider the formation and decomposition of a transition state as an example. I close by discussing the role of computational biochemistry in filling in those frames of our enzymatic movie that we cannot observe directly by time-resolved X-ray crystallography.

Picosecond Optical Second Harmonic Studies of Adsorbate Reduction Kinetics on Cadmium Sulfide

1993 ◽  
Vol 334 ◽  
Author(s):  
T. W. Scott ◽  
J. Martorell ◽  
Y. J. Chang
Keyword(s):  
Cadmium Sulfide ◽  
Near Infrared ◽  
Transient Absorption ◽  
Second Harmonic ◽  
Harmonic Signal ◽  
Recombination Luminescence ◽  
Kinetic Measurements ◽  
Time Resolved ◽  
Sum Frequency ◽  
Kinetics Of

AbstractTime resolved surface second harmonic generation has been used to probe the photoreduction kinetics of malachite green adsorbed onto single crystal cadmium sulfide. A detailed analysis is presented of how the adsorbates and the noncentrosymmetric substrate contribute separately to the total second harmonic signal. Conditions under which the adsorbates can be cleanly detected are described. To complement kinetic measurements of adsorbate reduction, the time evolution of conduction band carriers was determined using sum frequency up conversion of the recombination luminescence. In addition, the formation and decay of surface trapped carriers was monitored using near infrared transient absorption. Comparing the time scale for photoreduction with the relaxation kinetics of mobile and trapped charge carriers indicates that short lived mobile carriers rather than longer lived surface trapped carriers dominate interfacial charge transfer in this system.

scholarly journals Time-Resolved Infrared Spectroscopy of Binuclear Rhenium (I) Polypyridyl Complexes in Solution

1999 ◽  
Vol 19 (1-4) ◽  
pp. 279-282 ◽  
Author(s):  
L. C. Abbott ◽  
C. J. Arnold ◽  
K. C. Gordon ◽  
R. E. Hester ◽  
J. N. Moore ◽  
...  
Keyword(s):  
Mixed Valence ◽  
Difference Spectra ◽  
Kinetic Measurements ◽  
Time Resolved ◽  
Bridging Ligand ◽  
Ligand Charge Transfer ◽  
Complex Forms ◽  
Time Resolved Infrared Spectroscopy ◽  
State Kinetic ◽  
Mlct State

A series of four binuclear rhenium (I) complexes of the general form [Re(CO)3Cl]2BL, where BL is a polypyridyl bridging ligand, have been studied using ultrafast time-resolved UV/visible (TRVIS) and infrared (TRIR) spectroscopies. Visible excitation produces a metal-to-ligand charge-transfer (MLCT) excited state. Kinetic measurements show that the lifetime of this MLCT state varies between 100 and 1900 ps, depending on the structure of the bridging ligand. TRIR difference spectra show that each complex forms a similar MLCT state which has mixed valence character.

scholarly journals Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

2005 ◽  
Vol 280 (21) ◽  
pp. 20824-20832 ◽  
Author(s):  
Yoshihiro Yamaguchi ◽  
Takahiro Kuroki ◽  
Hisami Yasuzawa ◽  
Toshihiro Higashi ◽  
Wanchun Jin ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography

Selective removal of alpha heavy-chain glycosylation sites causes immunoglobulin A degradation and reduced secretion

1988 ◽  
Vol 8 (10) ◽  
pp. 4197-4203
Author(s):  
A K Taylor ◽  
R Wall
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin A ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Myeloma Cells ◽  
Heavy Chains ◽  
Attachment Sites ◽  
Glycosylation Sites ◽  
Kinetics Of

The importance of carbohydrate in the secretion of immunoglobulin A (IgA) has previously been suggested by results of studies with tunicamycin, which prevents N-linked glycosylation of all cell glycoproteins. To directly evaluate the role of individual oligosaccharides in the secretion of IgA, we have used site-directed mutagenesis to selectively eliminate the two N-linked attachment sites reported to be glycosylated in alpha heavy chains. Transfected wild-type and mutant alpha genes were expressed in kappa light-chain-producing MPC-11 variant myeloma cells, and secretion kinetics of the IgAs were compared. Removal of either or both glycosylation sites led to intracellular alpha heavy-chain degradation and a 90 to 95% inhibition of IgA secretion. These results reveal that both N-linked oligosaccharides of the alpha heavy chain are essential for intracellular stability and normal secretion of IgA. This suggests that the key function of carbohydrate here is to maintain proper conformation of the glycoprotein. We also found that when expressed in the MPC-11 variant cells, alpha heavy chains were glycosylated at a third, normally unused site.

scholarly journals Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics

2020 ◽  
Vol 477 (11) ◽  
pp. 2095-2114
Author(s):  
Rosario A. Muñoz-Clares ◽  
Lilian González-Segura ◽  
Javier Andrés Juárez-Díaz ◽  
Carlos Mújica-Jiménez
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxylase ◽  
Site Directed Mutagenesis ◽  
Allosteric Site ◽  
Allosteric Activation ◽  
Physiological Relevance ◽  
Phosphorylated Compounds ◽  
In The Wild ◽  
Kinetics Of

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation—in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)—was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239—which participates in a web of interactions of the protein with G6P—was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

Unprecedented Role of The N73-F124 Pair in The Staphylococcus equorum MnSOD Activity

2020 ◽  
Vol 16 ◽  
Author(s):  
Debbie Soefie Retnoningrum ◽  
Hiromi Yoshida ◽  
Muthia Dzaky Razani ◽  
Vincencius Felix Meidianto ◽  
Andrian Hartanto ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Manganese Superoxide Dismutase ◽  
Enzyme Stability ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Staphylococcus Equorum ◽  
The Stability

Background:: Bacterial manganese superoxide dismutase (MnSOD) occurs as a dimer, which is responsible for its activity and stability. Thereby, increasing the dimeric strength would increase the enzyme stability while maintaining its activity. Objective:: An N73F substitution was introduced to strengthen interactions between the monomers at the dimer interface. This substitution would introduce a π-stacking interaction between F73 of one monomer to F124 from the other monomer. Method:: Site-directed mutagenesis was carried out to substitute N73 with phenylalanine. The activity of the mutant was qualitative- and quantitatively checked while the stability was evaluated with a fluorescence-based thermal-shift assay. Finally, the structure of the mutant was elucidated by means of X-ray crystallography. Results:: The N73F mutant activity was only ~40% of the wildtype. The N73F mutant showed one TM at 60+1oC while the wildtype has two (at 52-55oC and 63-67oC). The crystal structure of the mutant showed the interactions between F73 from one monomer to F124 from the other monomer. The N73F structure presents an enigma because of no change in the enzyme structure including the active site. Furthermore, N73 and F124 position and interaction are conserved in human MnSOD but with a different location in the amino acid sequence. N73 has a role in the enzyme activity that is likely related to its interaction with F124, which resides in the active site region but has not been considered to participate in the reaction. Conclusion:: The N73F substitution has revealed the unprecedented role of the N73-F124 pair in the enzyme activity.

scholarly journals The role of lysine-67 in a class C β-lactamase is mainly electrostatic

1994 ◽  
Vol 302 (1) ◽  
pp. 1-4 ◽  
Author(s):  
D Monnaie ◽  
A Dubus ◽  
J M Frère
Keyword(s):  
Transient State ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Beta Lactam ◽  
Wild Type Enzyme ◽  
Individual Values ◽  
Class C ◽  
The Individual ◽  
State Kinetic

By using site-directed mutagenesis, the conserved Lys-67 residue situated three positions after the active-site Ser of a class C beta-lactamase was replaced by Arg or Gln. The Lys-67-Gln protein was nearly inactive. Although severely impaired, the Lys-67-Arg mutant exhibited an appreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the kcat. values were even increased. The properties of the Lys-67-Arg mutant were studied by both steady-state and transient-state kinetic methods with a variety of compounds representing distinct classes of available substrates. With beta-lactam substrates, the kcat./Km values reflecting the efficiency of the acylation step (k+2/K) were decreased 25-100-fold. When the individual values could be measured, k+2 was not significantly altered, but K was found to be strongly increased, a result most likely explained by a corresponding increase in the k+1/k-1 ratio. These results, combined with the much stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of the Lys-67 side chain.

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