Enzyme-substrate intermediates, problem

All these results are encouraging for investigators planning to use X-ray diffraction in mixed solvents at subzero temperatures and the rest of the present article will be devoted to a discussion of methods and preliminary results in this field. The methodology for cryoprotection of protein crystals, its physical-chemical basis, and the specific problems raised by the crystalline state, as well as the devices used to collect data at subzero temperatures, will be described. Limitations and perspectives of the procedure will be discussed critically. First attempts to determine the structure of productive enzyme-substrate intermediates through stop-action pictures will be described, as well as investigations showing that X-ray diffraction at selected normal and subzero temperatures can reveal protein structural dynamics. 

Work in solution is an absolute prerequisite for further studies of enzyme-substrate intermediates in the crystalline state. According to the Arrhenius relationship, k = A exp(—E IRT), which relates the rate constant k to the temperature, reactions normally occurring in the second to minute ranges might be sufficiently decreased in rate at subzero temperatures to permit intermediates to be stabilized, and occasionally purified by column chromatography if reactions are carried out in fluid solvent mixtures. Therefore, the first problem is to find a suitable cryoprotective solvent for the protein in question. 

The calculation of rate constants from steady state kinetics and the determination of binding stoichiometries requires a knowledge of the concentration of active sites in the enzyme. It is not sufficient to calculate this specific concentration value from the relative molecular mass of the protein and its concentration, since isolated enzymes are not always 100% pure. This problem has been overcome by the introduction of the technique of active-site titration, a combination of steady state and pre-steady state kinetics whereby the concentration of active enzyme is related to an initial burst of product formation. This type of situation occurs when an enzyme-bound intermediate accumulates during the reaction. The first mole of substrate rapidly reacts with the enzyme to form stoichiometric amounts of the enzyme-bound intermediate and product, but then the subsequent reaction is slow since it depends on the slow breakdown of the intermediate to release free enzyme. 

The problem can be handled using either the equilibrium approximation on the steady state approximation. Experiment shows, however, that true equilibrium is not achieved in the fast step because, the subsequent slow reaction is constantly removing the intermediate enzyme-substrate complex, ES. Generally, the enzyme concentration is far less than the substrate concentration, i.e., [E] [S], so that [ES] [S]. Hence, we can use the steady state approximation for the intermediate, ES. 

The elucidation of so many structures has allowed a successful classification of protein structures [7,8] it has laid the basis for certain predictive methods [9,10], and it has given insight into the possible evolutionary origins of proteins [11-13]. Our understanding of biological function in terms of structure has not increased so fast. This has turned out to be a more difficult problem. In the case of enzymes, a description of several different states of the protein complexed with substrate, intermediates and products together with necessary co-factors and activators is required. Often these different states can only be achieved by co-crystallisation, and even then it may be difficult to trap the necessary conformation. To date, it is... 

For the purpose of the present discussion the term transient kinetics is applied to the time course of a reaction from the moment when enzyme and substrate are mixed, t=0, until either a steady state or equilibrium is established. The difference between the kinetic problems discussed in section 3.3 and in the present section is, respectively, the presence of catalytic as distinct from catalytic concentrations of enzyme. Here we are concerned with the stoichiometry of enzyme states. Transient kinetic experiments with enzymes can be divided into two types. The first of these (multiple turnover) is carried out under the condition that the initial concentrations of substrate and enzyme are Cs(0) Ce(0) and c it) can, therefore, be regarded as constant throughout the course of the reaction until a steady state is attained. Alternatively, in a single turnover reaction, when Cs(0)reaction intermediates is observed until the overall process is essentially complete. These two possibilities will be illustrated with specific examples. In connection with a discussion of the approach to the steady state, in section 3.3 it was emphasized that, at t = 0, the concentrations of the intermediates, enzyme-substrate and enzyme-product complexes, are zero and, therefore, the rate of product formation is also zero. Under the experimental conditions used for steady state rate measurements and for enzyme assays, the first few seconds after the initiation of a reaction are ignored. However, when the experimental techniques and interpretation discussed below are used, events during the first few milliseconds of a reaction can be analysed and provide important information. With suitable monitors it is possible to follow the formation and decay of enzyme complexes with substrates and... 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

Such considerations raise the concept of the intrinsic kinetic isotope effect—the effect of isotopic substitution on a specific step in an enzyme-catalyzed reaction. The magnitude of an intrinsic isotope effect may not equal the magnitude of an isotope effect on collective rate parameters such as Vmax or Emax/ m, unless the isotopi-cally sensitive step is the rate-limiting or rate-contributing step. To tackle this problem, Northrop extended the kinetic theory for primary isotope effects in enzyme-catalyzed reactions. His approach can be illustrated with the following example of a one-substrate/two-intermedi-ate enzyme-catalyzed reaction ... 

To overcome problems of poor acceptor substrate acceptance, high concentrations of aldehyde substrates are required to obtain synthetically useful product yields. Unfortunately, DERA shows rather poor resistance to such high aldehyde concentrations, especially toward CIAA, resulting in rapid, irreversible inactivation of the enzyme. Therefore, the organic synthesis of (3R,5S)-6-chloro-2,4,6-trideoxy-hexapyranoside 1 requires very high amounts of DERA. Thus, despite the synthetic usefulness of DERA to produce chiral intermediates for statin side chains, the large-scale application is seriously hampered by its poor stability at industrially relevant aldehyde concentrations. The production capacity for such 2,4,6-trideoxy-hexoses of wild-type E. coli DERA is rather low [15]. 

The accumulation problem is most severe for enzymes such as the digestive enzymes, which have to cope with pulses of high substrate concentrations. If the concentration of the substrate is below the KM for the reaction under physiological concentrations, no intermediate accumulates in vivo in any case, since the enzyme is unbound. But in a test tube experiment in which the experimenter can use artificially high concentrations of substrate, an intermediate can sometimes be made to accumulate. An example occurs with glyceraldehyde 3-phosphate dehydrogenase. As Table 12.4 shows, the concentration of the aldehyde is below the Km in vivo. But in the laboratory, the acylenzyme accumulates at saturating substrate concentrations. 

Titration of the intact active site obviates problems due to inactive protein which contribute to a false molarity. Active-site titrations of acyl group transfer enzymes such as a-chymotrypsin utilise a substrate which has a good leaving group. This enables the buildup of an acyl enzyme intermediate which forms faster than it can degrade and results in... 

Molecular models show that during the course of the acylation reaction, the bound substrate is pulled partially out of the cyclodextrin cavity in forming the tetrahedral reaction intermediate. In other words, the model enzyme is not exhibiting the required transition state selectivity. Furthermore, excessively rigid substrates experience difficulty in rotating while bound in order to accommodate the need of the cyclodextrin hydroxyl group to attach perpendicular to the substrate ester plane, and subsequently rotate to become incorporated into the plane of the new ester product (Scheme 12.1). These problems were addressed by examination of substrates, such as p-nitro derivatives in which the ester protrudes further from the cavity, and substrates with more rotational flexibility such as alkyne 12.3. In these refined systems, much more enzyme-like rate accelerations of factors of up to 5 900 000-fold were observed for 12.4, for example. 

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