Charles Grubmeyer

L-histidinol dehydrogenase, a Zn2+-metalloenzyme.

The enzymatic activity of L-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 mM MnCl2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn2+/mol dimer, KD = 37 microM. ZnCl2, which prevented the MnCl2 stimulation, also bound to the enzyme, 1.2 g-atom/mol dimer, KD = 51 microM, and prevented Mn2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 M urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl2 or ZnCl2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl2 or ZnCl2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn2+, but which may bind Mn2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn2+ fulfills an essential structural and/or catalytic role.

Roles for cationic residues at the quinolinic acid binding site of quinolinate phosphoribosyltransferase.

Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) forms nicotinate mononucleotide (NAMN) from quinolinic acid (QA) and 5-phosphoribosyl 1-pyrophosphate (PRPP). Previously determined crystal structures of QAPRTase.QA and QAPRTase.PA.PRPP complexes show positively charged residues (Arg118, Arg152, Arg175, Lys185, and His188) lining the QA binding site. To assess the roles of these residues in the Salmonella typhimurium QAPRTase reaction, they were individually mutated to alanine and the recombinant proteins overexpressed and purified from a recombineered Escherichia coli strain that lacks the QAPRTase gene. Gel filtration indicated that the mutations did not affect the dimeric aggregation state of the enzymes. Arg175 is critical for the QAPRTase reaction, and its mutation to alanine produced an inactive enzyme. The k(cat) values for R152A and K185A were reduced by 33-fold and 625-fold, and binding affinity of PRPP and QA to the enzymes decreased. R152A and K185A mutants displayed 116-fold and 83-fold increases in activity toward the normally inactive QA analogue, nicotinic acid (NA), indicating roles for these residues in defining the substrate specificity of QAPRTase. Moreover, K185A QAPRTase displayed a 300-fold higher k(cat)/K(m) for NA over the natural substrate QA. Pre-steady-state analysis of K185A with QA revealed a burst of nucleotide formation followed by a slower steady-state rate, unlike the linear kinetics of WT. Intriguingly, pre-steady-state analysis of K185A with NA produced a rapid but linear rate for NAMN formation. The result implies a critical role for Lys185 in the chemistry of the QAPRTase intermediate. Arg118 is an essential residue that reaches across the dimer interface. Mutation of Arg118 to alanine resulted in 5000-fold decrease in k(cat) value and a decrease in the binding affinity of QA and PRPP to R152A. Equimolar mixtures of R118A with inactive or virtually inactive mutants produced approximately 50% of the enzymatic activity of WT, establishing an interfacial role for Arg118 during catalysis.

Interactions at the 2 and 5 Positions of 5-Phosphoribosyl Pyrophosphate Are Essential in Salmonella typhimurium Quinolinate Phosphoribosyltransferase

Quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) catalyzes an unusual phosphoribosyl transfer that is linked to a decarboxylation reaction to form the NAD precursor nicotinate mononucleotide, carbon dioxide, and pyrophosphate from quinolinic acid (QA) and 5-phosphoribosyl 1-pyrophosphate (PRPP). Structural studies and sequence similarities with other PRTases have implicated Glu214, Asp235, Lys153, and Lys284 in contributing to catalysis through direct interaction with PRPP. The four residues were substituted by site-directed mutagenesis. A nadC deletant form of BL21DE3 was created to eliminate trace contamination by chromosomal QAPRTase. The mutant enzymes were readily purified and retained their dimeric aggregation state on gel filtration. Substitution of Lys153 with Ala resulted in an inactive enzyme, indicating its essential nature. Mutation of Glu214 to Ala or Asp caused at least a 4000-fold reduction in k(cat), with 10-fold increases in K(m) and K(D) values for PRPP. However, mutation of Glu214 to Gln had only modest effects on ligand binding and catalysis. pH profiles indicated that the deprotonated form of a residue with pK(a) of 6.9 is essential for catalysis. The WT-like pH profile of the E214Q mutant indicated that Glu214 is not that residue. Mutation of Asp235 to Ala did not affect ligand binding or catalysis. Mutation of Lys284 to Ala decreased k(cat) by 30-fold and increased K(m) and K(D) values for PRPP by 80-fold and at least 20-fold, respectively. The study suggests that Lys153 is necessary for catalysis and important for PRPP binding, Glu214 provides a hydrogen bond necessary for catalysis but does not act as a base or electrostatically to stabilize the transition state, Lys284 is involved in PRPP binding, and Asp235 is not essential.

A Paradigm for Aldehyde Oxidation: Histidinol Dehydrogenase

Histidinol dehydrogenase (HDH, EC 1.1.1.23) catalyzes the oxidation of histidinol to histidine, using two moles of NAD. The reaction is the final step in the biosynthesis of histidine in bacteria, plants, and fungi. The enzyme is of particular interest in what it can tell us about dehydrogenase action: the reaction contains both alcohol and aldehyde dehydrogenase steps, apparently occurring at a single active site. Two other enzymes, UDP-glucose dehydrogenase (UDPGDH, EC 1.1.1.22) and hydroxymethyl glutaryl CoA reductase (HMGR, EC 1.1.1.34) catalyze conceptually similar 4-electron oxidations. Although the latter two have important roles in health and disease, for the enzymologist HDH offers the advantage of a long and interesting genetic history, and is particularly well suited to molecular approaches.

Probing the Nucleotide Binding Site of Creatine Kinasea

Creatine kinase (CPK) is a muscle enzyme that catalyzes the transfer of phosphoryl groups between the large reservoir of creatine phosphate and ADP to maintain the ATP pool used for muscle contraction. The enzymatic reaction is freely reversible. The dimeric rabbit muscle enzyme has been extensively studied,’ resulting in the identification of several of the amino acid residues at the nucleotide binding portion of the catalytic site. In addition, it has become clear that nucleotide binding and catalysis induce conformational changes in the enzyme that propagate unequally between the two subunits, resulting in structural asymmetry.’ To explore the role of nucleotide binding in catalysis, we have begun to employ 3’-~-(4-benzoyl)benzoyl ATP (BzATP),~ a newly synthesized photoaffinity analog of ATP. FIGURE 1 shows results of experiments designed to test BzATP as a substrate for the CPK reaction. A standard NADH-linked assay‘ was employed. Results indicated that BzATP was a substrate, with K , 0.23 mM and V , , , = 2.5 unit/mg. A parallel experiment with ATPshowed that K , = 0.4 mM and V,,, = 80 unit/mg. Thus BzATP appears to bind normally to the catalytic site, but shows the interesting property of being a “slow” substrate for the CPK reaction. This latter property may allow us to identify an individual reaction step at which the substitution at ribose hinders catalysis. BzATP was found to inactivate CPK when irradiated (filtered Hg arc) a t wavelengths above 320 nm. FIGURE 2 shows that this inactivation followed first order kinetics through at least 90% inhibition. Results also demonstrated that irradiation in the absence of BzATP did not produce appreciable inactivation. In separate experiments (not shown) we found that CPK incubated with BzATP for 30 rnin in the absence of light was not inhibited. To determine whether photoinactivation occurred at the catalytic site. substrate protection experiments were carried out. The presence of a “transition state analog complex”’ of ADP, creatine, KN03, and Mg2+ slowed the rate of inactivation about twofold (FIG. 2, curve C). The extent of the protection is not as large as expected from the tight binding of ADP in this complex. ADP alone causes BzATP inactivation kinetics to deviate from first order (not shown). Thus substrate protection experiments have not provided strong support for a catalytic site location for BzATP inactivation. An alternative approach for studying the interaction between BzATP and CPK is to study the concentration dependence of the inactivation rate. We have found (not shown) that inactivation of CPK by BzATP obeys saturation kinetics, indicating a two-step mechanism in which a noncovalent binding step precedes formation of the covalent bond. The Koba for the binding that leads to inactivation was 0.26 mM, very close to the K , for BzATP substrate binding.