Allosteric Effectors Regulate Certain Enzymes

Feedback inhibition refers to inhibition of an enzyme in a biosynthetic pathway by an end product of that pathway. For example, for the biosynthesis of D from A catalyzed by enzymes Enzj through Enz3,

Enz1

Enz2

Enz3

high concentrations of D inhibit conversion of A to B. Inhibition results not from the "backing up" of intermediates but from the ability of D to bind to and inhibit Enzj. Typically, D binds at an allosteric site spatially distinct from the catalytic site of the target enzyme. Feedback inhibitors thus are allosteric effectors and typically bear little or no structural similarity to the substrates of the enzymes they inhibit. In this example, the feedback inhibitor D acts as a negative allosteric effector of Enzj.

In a branched biosynthetic pathway, the initial reactions participate in the synthesis of several products. Figure 9-4 shows a hypothetical branched biosynthetic pathway in which curved arrows lead from feedback inhibitors to the enzymes whose activity they inhibit. The sequences S3 ^ A, S4 ^ B, S4 ^ C, and S3 ^ ^ D each represent linear reaction sequences that are feedback-inhibited by their end products. The pathways of nucleotide biosynthesis (Chapter 34) provide specific examples.

The kinetics of feedback inhibition may be competitive, noncompetitive, partially competitive, or mixed. Feedback inhibitors, which frequently are the small molecule building blocks of macromolecules (eg, amino acids for proteins, nucleotides for nucleic acids), typically inhibit the first committed step in a particular biosynthetic sequence. A much-studied example is inhibition of bacterial aspartate transcarbamoylase by CTP (see below and Chapter 34).

Multiple feedback loops can provide additional fine control. For example, as shown in Figure 9-5, the presence of excess product B decreases the requirement for substrate S2. However, S2 is also required for synthesis of A, C, and D. Excess B should therefore also curtail synthesis of all four end products. To circumvent this potential difficulty, each end product typically only partially inhibits catalytic activity. The effect of an excess of two or more end products may be strictly additive or, alternatively, may be greater than their individual effect (cooperative feedback inhibition).

Aspartate Transcarbamoylase Is a Model Allosteric Enzyme

Aspartate transcarbamoylase (ATCase), the catalyst for the first reaction unique to pyrimidine biosynthesis (Figure 34-7), is feedback-inhibited by cytidine tri-

Figure 9-4. Sites of feedback inhibition in a branched biosynthetic pathway. S,-S5 are intermediates in the biosynthesis of end products A-D. Straight arrows represent enzymes catalyzing the indicated conversions. Curved arrows represent feedback loops and indicate sites of feedback inhibition by specific end products.

Figure 9-4. Sites of feedback inhibition in a branched biosynthetic pathway. S,-S5 are intermediates in the biosynthesis of end products A-D. Straight arrows represent enzymes catalyzing the indicated conversions. Curved arrows represent feedback loops and indicate sites of feedback inhibition by specific end products.

Figure 9-5. Multiple feedback inhibition in a branched biosynthetic pathway. Superimposed on simple feedback loops (dashed, curved arrows) are multiple feedback loops (solid, curved arrows) that regulate enzymes common to biosynthesis of several end products.

phosphate (CTP). Following treatment with mercurials, ATCase loses its sensitivity to inhibition by CTP but retains its full activity for synthesis of carbamoyl as-partate. This suggests that CTP is bound at a different (allosteric) site from either substrate. ATCase consists of multiple catalytic and regulatory subunits. Each catalytic subunit contains four aspartate (substrate) sites and each regulatory subunit at least two CTP (regulatory) sites (Chapter 34).

Allosteric & Catalytic Sites Are Spatially Distinct

The lack of structural similarity between a feedback inhibitor and the substrate for the enzyme whose activity it regulates suggests that these effectors are not isosteric with a substrate but allosteric ("occupy another space"). Jacques Monod therefore proposed the existence of allosteric sites that are physically distinct from the catalytic site. Allosteric enzymes thus are those whose activity at the active site may be modulated by the presence of effectors at an allosteric site. This hypothesis has been confirmed by many lines of evidence, including x-ray crystallography and site-directed muta-genesis, demonstrating the existence of spatially distinct active and allosteric sites on a variety of enzymes.

Allosteric Effects May Be on Km or on Vmax

To refer to the kinetics of allosteric inhibition as "competitive" or "noncompetitive" with substrate carries misleading mechanistic implications. We refer instead to two classes of regulated enzymes: K-series and V-se-ries enzymes. For K-series allosteric enzymes, the substrate saturation kinetics are competitive in the sense that Km is raised without an effect on V^. For V-series allosteric enzymes, the allosteric inhibitor lowers Vmax

without affecting the Km. Alterations in Km or Vmax probably result from conformational changes at the catalytic site induced by binding of the allosteric effector at the allosteric site. For a K-series allosteric enzyme, this conformational change may weaken the bonds between substrate and substrate-binding residues. For a V-series allosteric enzyme, the primary effect may be to alter the orientation or charge of catalytic residues, lowering Vmax. Intermediate effects on Km and Vmax, however, may be observed consequent to these conforma-tional changes.

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