scholarly journals Reaction Initiation in Enzyme Crystals by Diffusion of Substrate

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 116 ◽  
Author(s):  
Marius Schmidt
Keyword(s):  
Ligand Binding ◽  
Large Body ◽  
X Ray ◽  
Substrate Complex ◽  
Catalytic Function ◽  
Enzyme Substrate ◽  
Sequence Of Events ◽  
Rate Limiting ◽  
Reaction Initiation ◽  
Key Questions

Ever since the first structure of an enzyme, lysozyme, was solved, scientists have been eager to explore how these molecules perform their catalytic function. There has been an overwhelmingly large body of publications that report the X-ray structures of enzymes determined after substrate and ligand binding. None of them truly show the structures of an enzyme working freely through a sequence of events that range from the formation of the enzyme–substrate complex to the dissociation of the product. The technical difficulties were too severe. By 1969, Sluyterman and de Graaf had pointed out that there might be a way to start a reaction in an enzyme crystal by diffusion and following its catalytic cycle in its entirety with crystallographic methods. The crystal only has to be thin enough so that the diffusion is not rate limiting. Of course, the key questions are as follows: How thin should the crystal be? Will the existing X-ray sources be able to collect data from a thin enough crystal fast enough? This review shines light on these questions.

Antithrombin - Mechanism Of Action And Binding Of Heparin

1981 ◽  
Author(s):  
I Björk ◽  
U Lindahl
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inactivation ◽  
Substrate Complex ◽  
High Affinity ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Rate Limiting

Antithrombin inhibits a variety of serine proteases by forming equimolar, inactive complexes with the enzymes. The anti thrombin-thrombin complex, extensively studied as a model for complexes with other coagulation proteases, dissociates with a half-life of several days to free enzyme and a proteolytically modified inhibitor. It thus behaves like a kinetically stable enzyme-substrate complex. Several observations indicate that deacylation is the rate-limiting step. The active site of antithrombin, i.e. the bond slowly cleaved by the target enzyme, is the Arg-385/Ser-386 bond in the carboxy-terminal region of the protein. The formation of most anti thrombin-protease complexes is greatly accelerated by certain forms of heparin. These active molecules comprise about 1/3 of normal heparin preparations and bind with high affinity (K∼108 M-1) to the inhibitor, regardless of the size of the polysaccharide. The stoichiometry of binding is 1:1 for most heparin molecules, although some high-molecular-weight chains have two antithrombin binding sites. Evidence from spectroscopic and kinetic analyses suggests that the binding of high-affinity heparin induces a conformational change in antithrombin that probably is involved in the mechanism of the increased rate of enzyme inactivation. Oligosaccharides with high-affinity for anti thrombin have been isolated by affinity chromatography following partial deaminative cleavage of heparin with nitrous acid. The smallest such oligosaccharide obtained is an octasaccharide, in which a pentasaccharide sequence appears to comprize the actual antithrombin-binding site. This active sequence contains a unique, 3-O-sulfated glucosamine residue that does not appear to occur in other portions of the heparin molecule. In addition, two N-sulfate groups and probably at least one O-sulfate group within the pentasaccharide sequence are essential for high-affinity binding of heparin to antithrombin.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

Kinetic Studies of the Flagellar Movement of Sea-Urchin Spermatozoa

1969 ◽  
Vol 50 (1) ◽  
pp. 203-222
Author(s):  
M. E. J. HOLWILL
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Sea Urchin ◽  
Activation Parameters ◽  
Kinetic Studies ◽  
Basic Part ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycerinated Model ◽  
Rate Limiting

1. The movements of intact and glycerinated spermatozoa from two species of sea-urchin were examined in the range of temperature between 5 and 30° C. The variations of the frequency of the glycerinated spermatozoa with changing pH, dielectric constant and ionic strength were examined. 2. From the temperature studies values for activation entropies and enthalpies were obtained that pertain to the chemical reaction which limits the frequency. 3. Within the limits of experimental error the activation parameters are identical for the intact spermatozoa and for the glycerinated model of the same species. The results are consistent with the hypothesis that the rate-limiting reaction is the breakdown of an enzyme-substrate complex. 4. The pH studies suggest that neither the acidic nor the basic part of the enzyme is involved in complex formation but that both participate in the breakdown of the complex. 5. The results obtained from the studies of dielectric constant are interpreted in terms of a model for the breakdown of the enzyme-substrate complex involving the separation of several spherical charges. 6. The studies of pH and ionic strength suggest that both 14 S and 30 S dynein participate in the mechano-chemical process responsible for bending the flagellum.

scholarly journals Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km

1976 ◽  
Vol 153 (2) ◽  
pp. 455-461 ◽  
Author(s):  
A Cornish-Bowden
Keyword(s):  
Dissociation Constants ◽  
Michaelis Constant ◽  
Ph Profile ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Catalytic Constant ◽  
Almost All ◽  
Rate Limiting

If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.

scholarly journals Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

2020 ◽  
Author(s):  
Suraj Pandey ◽  
George Calvey ◽  
Andrea M. Katz ◽  
Tek Narsingh Malla ◽  
Faisal H. M. Koua ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Ligand Binding ◽  
Direct Observation ◽  
Time Resolution ◽  
Data Set ◽  
X Ray ◽  
Substrate Complex ◽  
Millisecond Time Scale ◽  
Pulse Structure ◽  
Catalyzed Reactions

AbstractIn this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis β-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the ‘mix-and-inject’ technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of “molecular movies” which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance.One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

scholarly journals Catalytic properties of alkaline phosphatase from pig kidney

1974 ◽  
Vol 141 (1) ◽  
pp. 283-291 ◽  
Author(s):  
Kunio Hiwada ◽  
Ernst D. Wachsmuth
Keyword(s):  
Alkaline Phosphatase ◽  
Enzymic Activity ◽  
Ph Optimum ◽  
Active Centre ◽  
Substrate Complex ◽  
Pig Kidney ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (Vmax.) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme–substrate complex is 8.7 for p-nitrophenyl phosphate and β-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme–substrate complex, 8.7, is very similar to that for the enzyme–substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (ΔH) at pH9.5 showed a transition at 24.8°C that was unaffected by Mg2+. 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn2+ ions/tetrameric enzyme for an ordered association of the monomers. Zn2+ in excess and other bivalent ions compete for a second site with Mg2+. Mg2+ enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.

scholarly journals Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme–substrate complex

1999 ◽  
Vol 341 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Jaswir BASRAN ◽  
Michael J. SUTCLIFFE ◽  
Russ HILLE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Active Site ◽  
Bond Cleavage ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Trimethylamine Dehydrogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
Rate Limiting ◽  
Macroscopic Pka

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172 Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a ‘carbanion’ mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5±0.2 and 8.4±0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4±0.1 and 10.5±0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4±0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

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