Henrik Kacser

Evolution of catalytic proteins ― or: On the origin of enzyme species by means of natural selection

SummaryIt is believed that all present-day organisms descended from a common cellular ancestor. Such a cell must have evolved from more primitive and simpler precursors, but neither their organization nor the route such evolution took are accessible to the molecular techniques available today. We propose a mechanism, based on functional properties of enzymes and the kinetics of growth, which allows us to reconstruct the general course of early enzyme evolution. A precursor cell containing very few multifunctional enzymes with low catalytic activities is shown to lead inevitably to descendants with a large number of differentiated monofunctional enzymes with high turnover numbers. Mutation and natural selection for faster growth are shown to be the only conditions necessary for such a change to have occurred.

Studies on the antigens of Paramecium aurelia with the aid of fluorescent antibodies.

SUMMARY: Preparations of Paramecium aurelia, which had been immobilized by placing the organisms in a solution containing antiserum conjugated with fluorescein (conjugated antiserum), were examined by fluorescence microscopy. Unfixed paramecia accumulated fluorescent antibody in a thin layer around the entire surface of the organisms, and in globules at the clumped tips of the cilia, but not in the cilia themselves. No fluorescence was seen in the nuclei or the cytoplasm, but the food vacuoles became brightly fluorescent when the paramecia remained in conjugated antiserum for a few hours. Paramecia, which had been fixed with osmic acid and subsequently treated with fluorescent antibody, showed a faint fluorescence along the whole lengths of the cilia. When transformation from one serotype to another took place, the change in ability to take up a given kind of fluorescent antibody was seen to occur uniformly over the whole surface of the organism. It is concluded that the immobilization antigen is a fluid substance which covers the whole surface of the paramecia and is exuded into the medium under certain conditions, especially when homologous antibody is present.

Histidinaemia in mouse and man

A recently discovered mutant in the mouse was found to have very low levels of histidase. It is an autosomal recessive. In its enzymic and metabolic properties it appears to be a homologue of human histidinaemia. While the homozygous mouse mutants show no overt abnormalities, offspring of histidinaemic mothers display a balance defect resulting in circling behaviour. This is associated with vestibular damage during in utero development. Mental retardation caused by human maternal phenylketonuria may have a similar aetiology.

On Parts and Wholes in Metabolism

Historically, the progress of science has been driven by its analytical approach. Although the biological sciences have been lagging somewhat behind the physical sciences, the last fifty years or so have seen an increasing avalanche in the identification and description of the components of living organisms. Refined instrumentation and ingenious techniques have gone hand in hand with sophisticated mathematical models of molecular properties. The method of choice has been to cut up the organism into ever smaller pieces and look at the pieces in ever greater detail. We can now detect a single specific nucleotide among millions and measure the presence of a few molecules in a whole cell. We can describe the dynamics of internal molecular motions and calculate the thermodynamics of their transitions.

Control of the Flux in the Arginine Pathway of Neurospora crassa: Effects of Co-ordinate Changes of Enzyme Concentration

Summary: The flux to arginine was determined in growing mycelium of Neurospora crassa carrying the aga mutation, by measuring the exponential growth rate and the arginine content of the free pool and that in protein. Derepression of the enzymes in the pathway by a factor of about three resulted in a 14% increase in the flux. Using a mutant (cpc-1), which affected the cross-pathway control, the pathway enzymes were found to have about threefold lower activities. This resulted in a 16% decrease in the measured flux. The effect of arginine acting as a feedback inhibitor of acetylglutamate kinase was estimated in an arg-12 mutant by varying the steady-state arginine concentration using histidine as a competitive inhibitor of the citrulline uptake. It is concluded that the feedback loop is mainly responsible for the small response of the flux to the large coordinate changes in the pathway enzymes. The results are discussed in terms of control analysis of metabolic systems.

Comparison of the properties of histidine ammonia-lyase in normal and histidinemic mutant mice.

The histidinemic (his/his) mutant mouse shows greatly reduced skin and liver histidine:ammonia-lyase (HAL; EC 4.3.1.3) activity compared with normal mice. Liver HAL activity in the mutant is heat and salt labile and is inhibited at high substrate concentrations. Two HAL components have been identified in the normal mouse liver, a minor component with properties similar to those of HAL of the mutant mouse and a major component which is heat and salt stable and insensitive to substrate inhibition. Immunotitration with anti-HAL antibody shows that the livers of mutant mice contain no detectable antigenically cross-reacting HAL protein. It is concluded, therefore, that the his allele is a null allele at a structural or regulatory locus for the major HAL enzyme and maps close to the HAL-regulatory locus Hsd and that the low residual HAL activity in the mutant is due to another enzyme.

Control Analysis of Systems with Enzyme-Enzyme Interactions

The classical approach to control analysis has made two fundamental assumptions: the first is that all enzymes are independently acting catalysts, and the second is that the reaction rate of an isolated enzyme is first-order with respect to enzyme concentration. The first assumption, that of independence, implies that no direct interactions between enzymes affect their catalytic activities (other than that which occurs indirectly through the common substrate or effector pools). This assumption would be invalid, for example, if dynamic enzyme-complex formation affected the kinetic parameters of the constituent enzymes. The second assumption, that of additivity, based on classical enzymology, would not apply if, for example, an enzyme oligomer-monomer equilibrium existed for a single reaction step, or if the enzyme were partitioned between free and membrane-bound forms, the membrane being present in fixed amount, with different activities of the two forms.

Derepression of enzyme synthesis in response to arginine limitation in Neurospora crassa

Ornithine carbamoyltransferase and argininosuccinase, two enzymes involved in arginine synthesis, are regulated by cross-pathway amino acid control in Neurospora and show derepression in response to limitation of any one of a number of amino acids. The effects of varying the severity of arginine limitation upon the synthesis of these enzymes, in mycelial cultures of an arginine auxotrophic strain, are reported here. Depression occurred at arginine concentrations sufficient to allow normal rates of protein accumulation, leading to increases of not more than fourfold in the absolute rate of enzyme synthesis. On the other hand, differential rates of enzyme synthesis increased progressively up to 20-fold or more under extreme conditions of arginine limitation that also limit net protein synthesis. The major part of the derepression response thus occurred at arginine concentrations that allowed low net rates of protein synthesis. The physiological significance of this is not yet understood. Our evidence suggests that these responses were mediated entirely through the cross-pathway control system, and may not be untypical (allowing for variations in magnitude) of depression resulting through this mechanism in Neurospora.

Allosteric repression: An Analysis

Abstract A kinetic analysis of repression is presented based on the accepted sequence of molecular events and the assumption that the co-repressor-repressor interaction involves an allosteric transition. This leads to an expression which relates the two experimentally accessible variables—enzyme and signal—when the system is operating in vivo . A suitable plot allows the estimation of a coefficient, h max , which is related to the number of protomers, n , of the oligomeric repressor protein. This parameter is similar to but distinct from a Hill coefficient for allosteric inhibitions. Two examples from the literature are analysed in terms of the model.

Genetic effects on susceptibility to histidine induced teratogenesis in the mouse

In the mouse histidinaemia has a teratogenic effect. Animals subjected to high levels of histidine in utero may develop inner ear and behavioural abnormalities typical of the ‘shaker–waltzer’ syndrome. Selection procedures for abnormalities and relaxation of selection have resulted in two histidinaemic strains: the Cambridge strain in which abnormalities occur in over 80% of animals, and the Edinburgh strain in which the penetrance of abnormal behaviour has declined to about 1%. Breeding experiments suggest that the differences are largely due to differences in the genetic backgrounds which modify foetal susceptibilities to the teratogenic effects of high histidine levels. High susceptibility appears to be dominant over low susceptibility in the present strains. There appears to be no interaction of maternal histidinaemia with the dreher mutation which is considered to induce inner ear malformation as a result of an early neural tube abnormality.