Enzyme Kinetics —

When I first studied Biochemistry in RB Fisher in Edinburgh in the early 1960s I was drawn into his enthusiasm for enzyme kinetics. I was so ignorant and yet had a medical degree and was entranced by this topic. He was very mathematical in his approach.

I wondered at the time if there was but one chemical structure for a protein enzyme and that different biochemical processes used different sections of the structure. I was so obviously wrong. This was before the expansion of knowledge of structure and structure relationships that we now enjoy.

In Henzler-Wildman et al Nature 2007 Intrinsic motions along an enzymatic reaction trajectory vol 450 pp 838-44 there is a fascinating paper which uses a variety of analytical techniques to identify how a protein assumes a shape which maximises and facilitates enzyme function.

A folded protein is not a unique structure, but includes an ensemble of folded states at physiological temperatures.

Protein folding does not happen by random sampling of all possible conformation. The rearrangements within a folded protein are directed by the energy requirements. Although the lowest energy structures can often be determined experimentally, an understanding of other conformations and the transitions among them is still in its infancy.

A relationship between structure and freedom of movement and shape plasticity results in the unique power of biocatalysts (enzymes). The chemical mechanisms of many enzymatic reactions are known in great detail thanks to advances in classical enzymology and structural biology. For a number of enzymes, snapshots of conformations that are sampled during catalysis have been obtained using ligands, substrates and inhibitors. Recently, transitions between these states have been measured by nuclear magnetic resonance (NMR) relaxation experiments with substrate analogues or during catalysis, as well as by single-molecule fluorescence resonance energy transfer (FRET). In this paper the authors explore how an enzyme, adenylate kinase, reaches a catalytically competent conformation in which the reactive groups are brought into close proximity in a position favouring catalysis.

Using X-ray crystallography, NMR, single-molecule FRET, normal mode analysis (NMA) and molecular dynamics simulations, they identify1 conformational substates during a reaction.

The motions in the form of the protein enzyme are as one might anticipate, not random but follow a pathway which enables a configuration capable of effective catalytic activity.

Metabolism —

Metabolic pathways Reinhart Heinrich Nature 2006 447, p700

Pioneer in systems biology.

In biology, mathematical systems analysis was until recently nearly invisible in the dazzling light of twentieth-century discoveries. But it has emerged from the shadows in the field of systems biology, a subject buoyed by immense data sets, conveyed by heavy’ computing power, and addressing seemingly incomprehensible forms of complexity. If systems biology has heroes, one of them is Reinhart Heinrich, a former professor at the Humbotdt University in Berlin, who died on 23 October, aged 60. His most famous accomplishment was metabolic control theory, published in 1974 with Tom Rapoport and formulated independently by Henrik Kacser and James A. Burns in Edinburgh, UK.

From the 1930s to the 1960s, biochemists were busy describing metabolic pathways, just as molecular biologists today are feverishly trying to inventory the cell’s gene-transcription and signalling circuits. The basic kinetic features of the enzymes in the major pathways were studied in great detail and with exceeding care. It seemed self-evident that, knowing the properties of each element, the behaviour of a pathway at vivo could simply be understood as the sum of its enzymes.

One assertion, drilled into the head of every biochemist, was the concept of the rate-limiting step. In this view, the flux through a pathway was determined by the slowest reaction, in the way a bucket brigade fighting a fire would be limited by the speed of the slowest member. Yet this concept, as shown by the metabolic control theory, was theoretically flawed, practically incomplete and often wrong, as many efforts to genetically engineer enzymes by changing ‘rate-limiting steps’ would ultimately show.

Metabolic control theory introduced the concept of control coefficients dimensionless quantities indicating how the flux of a pathway depended on a given step. Only a pathway where every control coefficient except one was zero would have a rate-limiting step, since the flux of that pathway would depend only on that step. Several pathways in fact had rate-limiting steps, but that often reflected the structure of the pathway, and could not simply be deduced from the maximum rates of the individual enzymes, their Michaelis constants or their displacement from equilibrium. Many pathways were instead networks, with the fluxes distributed in a self-governing way among its various branches.

Heinrich went on to apply this theory to the real case of glycolysis in red blood cells, where he showed that the flux of the reaction was shared by several enzymes. Much later, he extended his ideas to signal-transduction pathways, introducing control coefficients to dynamic processes. Sticking to real examples, such as the Wnt signalling and MAP kinase pathways, he again demonstrated that new properties and constraints emerge when the individual steps are combined into a complete pathway.

Heinrich also pointed the way to considerations of optimality theory and evolution that will confront systems biology for the next century. The question of evolution lies just beneath any effort to understand biology. Yet in most cases, physiological function and evolutionary change are considered distinct and are investigated by different people. Heinrich’s work illustrated how systems biology might develop, where the central question will be not only how a system.

Metabolomics —

Metabolomics: an emerging post-genomic tool for nutrition

Metabolomics is the study of the raw materials and products of the body’s biochemical reactions. Metabolomics is concerned with the analysis and measurement of global sets of low-molecular-weight compounds in urine, blood or some other body fluid, scanned in a NMR spectroscopy or Mass Spectrometer and to provide a profile of tens or hundreds of chemicals that can predict whether an individual vulnerable to a disease, or may develop side-effects from a particular drug. Such profiles may provide a more comprehensive view of cellular control mechanisms in man and animals, and raise the possibility of identifying surrogate markers of disease. Researchers are already trying to identify whether a person will develop specific diseases by measuring levels of gene expression or proteins, but supporters of metabolomics say they should be able to do it better. Small changes in the activity of a gene or protein (which may have an unknown impact on the workings of a cell) often create a much larger change in metabolite levels. The approach has already proved its worth: cholesterol and glucose have long been chemical indicators for heart disease and diabetes. Metabolomics has been made possible by the development of technologies that allow the function of cells and whole organisms to be explored at the molecular level. metabolites

Metabolomics has already been used to study toxicological mechanisms and disease processes and offers enormous potential as a means of investigating the complex relationship between nutrition and metabolism. Examples include the metabolism of dietary substrates, drug-induced disturbances of lipid metabolites in type 2 diabetes mellitus and the therapeutic effects of vitamin supplementation in the treatment of chronic metabolic disorders.

But realising this vision isn’t straightforward. One of the first tasks is to create a catalogue of compounds in the human body, and this is proving hard to define. David Wishart at the University of Alberta, Edmonton, and his colleagues have taken an initial step forward by producing the first draft of the human metabolome. They searched the published literature for known human metabolites, and have collected around 2,500 of them into a public database www.hmdb.ca

However this may be an oversimplification. When the effect of age, gender, food physical makeup and chemical composition of diets rich in soya phyto-oestrogens on the absorption of the isoflavones was studied, urinary excretion of the metabolites was influenced by the isoflavone chemical make up of the diet , the sex of the person ingesting the food but not their age.

Nevertheless this is an exciting new area in Nutrition.

Terminology

Metabolomics The comprehensive analysis of the whole melabolome under a given set of conditions

Metabonomics: The quantitative measurement of time-related multiparametric metabolic responses of multicellular systems to pathophysiological stimuli or genetic modification

Metabolome The full set of low-molecular-weight metabolites within, or that can be secreted by, a given cell type or tissue

Metabolic fingerprinting The application of any technological approach whose output is processed with pattern-recognition software and without differentiation of individual metabolites

Lipidomics The characterisation of chemically distinct lipid species in cells and the molecular mechanisms through which they facilitate cellular function

Phillip D. Whitfield et al in British Journal of Nutrition 2004, 92, 549-555

Pearson H in Nature, 2007, 446, 8

Faughan MS et al in British Journal of Nutrition 2004, 91, 567-574

Amino Acid and Protein Digestion
Prion Detection Technique —

Prion infection is a worry with beef in the human food chain. This ratheresoteric paper is an important step in the detection of this threat to human well being.,

Privat et al 2008 Human prion diseases: from antibody screening to a standardized
fast immunodiagnosis using automation. Modern Pathology vol 21, 140-149

Demopstration of pathological prion protein accumulation in the central nervous system is required to establish the diagnosis of transmissible subacute encephalopathies. In humans, this is frequently achieved using prion protein immunohistochemistry in paraffin-embedded tissue, a technique that requires multiple epitope retrieval and denaturing pretreatments. In addition to being time-consuming this procedure induces tissue alterations that preclude accurate morphological examination. The aim of the study described in this paper was to simplify prion protein immunohistochemistry procedure in human tissue, together with increased sensitivity and specificity. A panel of 50 monoclonal antibodies were produced using various immunugens ( human and ovine recombinant prion protein, prion protein peptides. denatured scrapie associated fibrils from 263K-infected Syrian hamsters) and directed against different epitopes along the human prion protein sequence. A panel of different forms of genetic, infectious and sporadic transmissible subacute encephalopathies was assessed. The monoclonal 12F10 antibody provided a high specificity and fast immunodiagnosis with very limited denaturing pretreatments. A standardized and reliable fast imnmnostaining procedure was established using an automated diagnostic system and allowed prion protein detection in the- central nervous system and in tonsil biopsies. It was evaluated in a series of 300 patients with a suspected d iagnosis of transmissible subacute transmissible encephalopaties and showed high sensitivity.

Protein Turnover —

Protein turnover is the continual synthesis and breakdown of protein in the body. This is a core process in biology and many talents have attempted to measure this in the whole body .

The use of radio labelled amino acids requires a model system to enable calculations.

Q = I + B + N = S + M + C

Q is the flux of the amino acid, I the dietary intake, plus the input from protein breakdown B and N is the input from de novo synthesis. The flux is equal to the incorporation of the amino acid into body protein S, oxidation and other forms of metabolism M. There is small additional loss from the gastrointestinal tract as faeces

( 1 g N / day ) , skin ( 20 mg N / kg body weight ) and non measurable urinary uric acid ( 3 % of urinary nitrogen). Q can be calculated with the flux based on NH3 ( Q A ) or on urea (Q u ).

In human studies stable isotopes are used eg 13 C and 15 N. The amino acid chosen is usually leucine as this has one catabolic pathway and is predominantly metabolised in muscle. The amino acid can be given orally or more usually intravenously for 2 hours to achieve a plateau enrichment of the plasma. If leucine is used, its muscle metabolic product a -keto isocaproic acid (KIC) is used for measurement, being a better indicator of intracellular leucine enrichment.

Such methodology requires that a steady state plasma concentration of tracer is achieved
the dose of tracer has no consequences for the metabolism of the tracer.
the labelled and unlabelled amino acids are metabolised identically
no significant recyling of the isotope occurs
the enrichment in plasma is representative of that at the site of protein synthesis.
In a review Duggleby and Waterlow ( British Journal of Nutrition 2005, vol 94, pp 141-153 ) have analysed an cheap alternative, the end product approach using labelled glycine. This method has merit for population results.

This method relies on a two-pool model, and that the two products of the same precursor have the same labelled activity. The other assumption is that the activity of the end product reflects the amino acid-N mixture taken up into protein. The end products of ammonia or urea may not give the same result.

In the complex of all the proteins in the body and their varied rate of turnover this is a basic assumption, and is reflected in the range of inexplicable results.

If nitrogen flux is measured in the same subject over a period of time using a range of amino acid precursors then the measure of protein synthesis ( mg protein /kg per hour) ranges from 70 to 1038 ) and between oral and intravenous. However closer results are obtained from the individual amino acids. Few studies have been made on the same subject using different labelled amino acids at the same time.

In many respects this is an unresolved and demanding area.

Review Duggleby and Waterlow (British Journal of Nutrition 2005, vol 94, pp 141-153)