The final enzymes that we shall consider are the nucleoside monophosphate kinases (NMP kinases), typified by adenylate kinase. These enzymes catalyze the transfer of the terminal phosphoryl group from a nucleoside triphosphate (NTP), usually ATP, to the phosphoryl group on a nucleoside monophosphate (Figure 9.45). The challenge for NMP kinases is to promote the transfer of the phosphoryl group from NTP to NMP without promoting the competing reactionthe transfer of a phosphoryl group from NTP to water; that is, NTP hydrolysis. We shall see how the use of induced fit by these enzymes is used to solve this problem. Moreover, these enzymes employ metal ion catalysis; but, in this case, the metal forms a complex with the substrate to enhance enzyme-substrate interaction.

X-ray crystallographic methods have yielded the three-dimensional structures of a number of different NMP kinases, both free and bound to substrates or substrate analogs. Comparison of these structures reveals that these enzymes form a family of homologous proteins (Figure 9.46). In particular, such comparisons reveal the presence of a conserved NTP-binding domain. This domain consists of a central b sheet, surrounded on both sides by a helices (Figure 9.47). A characteristic feature of this domain is a loop between the first b strand and the first helix. This loop, which typically has an amino acid sequence of the form Gly-X-X-X-X-Gly-Lys, is often referred to as the P-loop because it interacts with phosphoryl groups on the bound nucleotide (Figure 9.48). As described in Section 9.4.4, similar domains containing P-loops are present in a wide variety of important nucleotide-binding proteins.

Kinetic studies of NMP kinases, as well as many other enzymes having ATP or other nucleoside triphosphates as a substrate, reveal that these enzymes are essentially inactive in the absence of divalent metal ions such as magnesium (Mg²⁺) or manganese (Mn²⁺), but acquire activity on the addition of these ions. In contrast with the enzymes discussed so far, the metal is not a component of the active site. Rather, nucleotides such as ATP bind these ions, and it is the metal ion-nucleotide complex that is the true substrate for the enzymes. The dissociation constant for the ATP-Mg²⁺ complex is approximately 0.1 mM, and thus, given that intracellular Mg²⁺ concentrations are typically in the millimolar range, essentially all nucleoside triphosphates are present as NTP-Mg²⁺ complexes.

How does the binding of the magnesium ion to the nucleotide affect catalysis? There are a number of related consequences, but all serve to enhance the specificity of the enzyme-substrate interactions by enhancing binding energy. First, the magnesium ion neutralizes some of the negative charges present on the polyphosphate chain, reducing nonspecific ionic interactions between the enzyme and the polyphosphate group of the nucleotide. Second, the interactions between the magnesium ion and the oxygen atoms in the phosphoryl group hold the nucleotide in well-defined conformations that can be specifically bound by the enzyme (Figure 9.49). Magnesium ions are typically coordinated to six groups in an octahedral arrangement. Typically, two oxygen atoms are directly coordinated to a magnesium ion, with the remaining coordination positions often occupied by water molecules. Oxygen atoms of the a and b, b and g, or a and g phosphoryl groups may contribute, depending on the particular enzyme. In addition, different stereoisomers are produced, depending on exactly which oxygen atoms bind to the metal ion. Third, the magnesium ion provides additional points of interaction between the ATP-Mg²⁺ complex and the enzyme, thus increasing the binding energy. In some cases, such as the DNA polymerases (Section 27.2.2), side chains (often aspartate and glutamate residues) of the enzyme can bind directly to the magnesium ion. In other cases, the enzyme interacts indirectly with the magnesium ion through hydrogen bonds to the coordinated water molecules (Figure 9.50). Such interactions have been observed in adenylate kinases bound to ATP analogs.

Comparison of the structure of adenylate kinase in the presence and absence of an ATP analog reveals that substrate binding induces large structural changes in the kinase, providing a classic example of the use of induced fit (Figure 9.51). The P-loop closes down on top of the polyphosphate chain, interacting most extensively with the b phosphoryl group. The movement of the P-loop permits the top domain of the enzyme to move down to form a lid over the bound nucleotide. This motion is favored by interactions between basic residues (conserved among the NMP kinases), the peptide backbone NH groups, and the nucleotide. With the ATP nucleotide held in this position, its g phosphoryl group is positioned next to the binding site for the second substrate, NMP. In sum, the direct interactions with the nucleotide substrate lead to local structural rearrangements (movement of the P-loop) within the enzyme, which in turn allow more extensive changes (the closing down of the top domain) to take place. The binding of the second substrate, NMP, induces additional conformational changes. Both sets of changes ensure that a catalytically competent conformation is formed only when both the donor and the acceptor are bound, preventing wasteful transfer of the phosphoryl group to water. The enzyme holds its two substrates close together and appropriately oriented to stabilize the transition state that leads to the transfer of a phosphoryl group from the ATP to the NMP. This is an example of catalysis by approximation. We will see such examples of a catalytically competent active site being generated only on substrate binding many times in our study of biochemistry.

Domains similar (and almost certainly homologous) to those found in NMP kinases are present in a remarkably wide array of proteins, many of which participate in essential biochemical processes. Examples include ATP synthase, the key enzyme responsible for ATP generation; molecular motor proteins such as myosin; signal-transduction proteins such as transducin; proteins essential for translating mRNA into proteins, such as elongation factor Tu; and DNA and RNA unwinding helicases. The wide utility of P-loop NTPase domains is perhaps best explained by their ability to undergo substantial conformational changes on nucleoside triphosphate binding and hydrolysis. We shall encounter these domains (hereafter referred to as P-loop NTPases) throughout the book and shall observe how they function as springs, motors, and clocks. To allow easy recognition of these domains, they, like the binding domains of the NMP kinases, will be depicted with the inner surfaces of the ribbons in a ribbon diagram shown in purple and the P-loop shown in green (Figure 9.52).