Ligand bound structure of a 6-hydroxynicotinic acid 3-monooxygenase provides mechanistic insights.

6-Hydroxynicotinic acid 3-monooxygenase (NicC) is a bacterial enzyme involved in the degradation of nicotinic acid. This enzyme is a Class A flavin-dependent monooxygenase that catalyzes a unique decarboxylative hydroxylation. The unliganded structure of this enzyme has previously been reported and studied using steady- and transient-state kinetics to support a comprehensive kinetic mechanism. Here we report the crystal structure of the H47Q NicC variant in both a ligand-bound (solved to 2.17 Å resolution) and unliganded (1.51 Å resolution) form. Interestingly, in the liganded form, H47Q NicC is bound to 2-mercaptopyridine (2-MP), a contaminant present in the commercial stock of 6-mercaptopyridine-3-carboxylic acid(6-MNA), a substrate analogue. 2-MP binds weakly to H47Q NicC and is not a substrate for the enzyme. Based on kinetic and thermodynamic characterization, we have fortuitously captured a catalytically inactive H47Q NicC•2-MP complex in our crystal structure. This complex reveals interesting mechanistic details about the reaction catalyzed by 6-hydroxynicotinic acid 3-monooxygenase.

Mechanism of the Multistep Catalytic Cycle of 6-Hydroxynicotinate 3-Monooxygenase Revealed by Global Kinetic Analysis.

The class A flavoenzyme 6-hydroxynicotinate 3-monooxygenase (NicC) catalyzes a rare decarboxylative hydroxylation reaction in the degradation of nicotinate by aerobic bacteria. While the structure and critical residues involved in catalysis have been reported, the mechanism of this multistep enzyme has yet to be determined. A kinetic understanding of the NicC mechanism would enable comparison to other phenolic hydroxylases and illuminate its bioengineering potential for remediation of N-heterocyclic aromatic compounds. Toward these goals, transient state kinetic analyses by stopped-flow spectrophotometry were utilized to follow rapid changes in flavoenzyme absorbance spectra during all three stages of NicC catalysis: (1) 6-HNA binding; (2) NADH binding and FAD reduction; and (3) O2 binding with C4a-adduct formation, substrate hydroxylation, and FAD regeneration. Global kinetic simulations by numeric integration were used to supplement analytical fitting of time-resolved data and establish a kinetic mechanism. Results indicate that 6-HNA binding is a two-step process that substantially increases the affinity of NicC for NADH and enables the formation of a charge-transfer-complex intermediate to enhance the rate of flavin reduction. Singular value decomposition of the time-resolved spectra during the reaction of the substrate-bound, reduced enzyme with dioxygen provides evidence for the involvement of C4a-hydroperoxy-flavin and C4a-hydroxy-flavin intermediates in NicC catalysis. Global analysis of the full kinetic mechanism suggests that steady-state catalytic turnover is partially limited by substrate hydroxylation and C4a-hydroxy-flavin dehydration to regenerate the flavoenzyme. Insights gleaned from the kinetic model and determined microscopic rate constants provide a fundamental basis for understanding NicC's substrate specificity and reactivity.

Early Intravenous Magnesium Sulfate Administration in the Emergency Department for Severe Asthma Exacerbations.

BACKGROUND: Severe asthma exacerbations in pediatric patients occur frequently and can require pediatric intensive care unit (PICU) admission. OBJECTIVE: To determine if early administration of intravenous magnesium sulfate (IVMg) to pediatric patients experiencing severe asthma exacerbations, defined as a respiratory clinical score (RCS) of 9 to 12, resulted in fewer PICU admissions. METHODS: Retrospective chart review of pediatric patients aged from 2 to 17 years presenting with a severe asthma exacerbation to a single tertiary care pediatric emergency department. Univariable and multivariable logistic regression analyses were used to determine if admission to the PICU was associated with early IVMg treatment, within 60 minutes of registration. RESULTS: A total of 1911 patients were included in the study, of which 1541 received IVMg. The average time to IVMg was 79 minutes, with 35% of the patients receiving it within 60 minutes of arrival. Two hundred forty-eight (13%) were admitted to the PICU, 641 (34%) were admitted to the general inpatient floor, and 1022 (53%) were discharged home. Factors associated with increased odds ratio (OR) of PICU admission were: early IVMg (OR, 1.63; 95% CI: 1.16-2.28), arrival mode to the emergency department via ambulance (OR, 2.23; 95% CI: 1.45-3.43), history of PICU admission for asthma (OR, 1.73; 95% CI: 1.22-2.44), and diagnosis of status asthmaticus (OR, 8.88; 95% CI: 3.49-30.07). Calculated OR of PICU admission subcategorized by RCS for early IVMg patients, after controlling for PICU risk factors, are as follows: RCS 9 (reference), RCS 10 (OR, 2.52; 95% CI: 0.89-2.23), RCS 11 (OR, 2.19; 95% CI: 1.3-3.70), and RCS 12 (OR, 4.12; 95% CI: 2.13-7.95). CONCLUSIONS: Early administration of IVMg to pediatric patients experiencing severe asthma exacerbations does not result in fewer PICU admissions.

Synthesis and crystallographic characterization of 6-hydroxy-1,2-dihydropyridin-2-one.

The title compound, C5H5NO2, is a hy-droxy-lated pyridine ring that has been studied for its involvement in microbial degradation of nicotinic acid. Here we describe its synthesis as a formic acid salt, rather than the standard hydro-chloride salt that is commercially available, and its spectroscopic and crystallographic characterization.

Human matters in asthma: Considering the microbiome in pulmonary health.

Microbial communities form an important symbiotic ecosystem within humans and have direct effects on health and well-being. Numerous exogenous factors including airborne triggers, diet, and drugs impact these established, but fragile communities across the human lifespan. Crosstalk between the mucosal microbiota and the immune system as well as the gut-lung axis have direct correlations to immune bias that may promote chronic diseases like asthma. Asthma initiation and pathogenesis are multifaceted and complex with input from genetic, epigenetic, and environmental components. In this review, we summarize and discuss the role of the airway microbiome in asthma, and how the environment, diet and therapeutics impact this low biomass community of microorganisms. We also focus this review on the pediatric and Black populations as high-risk groups requiring special attention, emphasizing that the whole patient must be considered during treatment. Although new culture-independent techniques have been developed and are more accessible to researchers, the exact contribution the airway microbiome makes in asthma pathogenesis is not well understood. Understanding how the airway microbiome, as a living entity in the respiratory tract, participates in lung immunity during the development and progression of asthma may lead to critical new treatments for asthma, including population-targeted interventions, or even more effective administration of currently available therapeutics.

The Safety of High-Dose Intranasal Fentanyl in the Pediatric Emergency Department.

OBJECTIVES: This study aimed to determine if the use of intranasal (IN) fentanyl in the pediatric emergency department of 2 to 5 µg/kg at doses greater than 100 µg is associated with adverse events in pediatric patients. METHODS: We performed a retrospective chart review of patients receiving IN fentanyl at an urban, tertiary care emergency department in Memphis, TN, from January 1, 2011, to December 31, 2017. All adverse events documented through the hospital's voluntary safety reporting system involving IN fentanyl were reviewed to determine patient outcomes. RESULTS: A total of 3205 patients received greater than 100 µg of IN fentanyl during the study period from 2011 to 2017. The average (SD) patient age was 13.7 (2.65) years, ranging from 5 to 18 years. The mean (SD) initial dose was 162 (30) µg ranging from 102 to 265 µg (2 doses were given greater than 200 µg in the study period). Initial average (SD) dose for weight was 2.62 (0.5) µg/kg. A total of 13 adverse events were documented, with only 3 occurring at doses greater than 100 µg. No patients required the reversal agent naloxone or invasive respiratory support. CONCLUSIONS: To our knowledge, this is the first study using doses greater than 100 µg of IN fentanyl in a pediatric population. Our results indicate that fentanyl can be safely administered at doses of greater than 100 µg without any clinically significant adverse outcomes observed for 7 years of use. It is our hope that this information will increase utilization of IN fentanyl for treatment of acute pain in emergency departments and in the prehospital setting.

The Impact of Terbutaline as Adjuvant Therapy in the Treatment of Severe Asthma in the Pediatric Emergency Department.

METHODS: Patients were identified using a retrospective cohort analysis from a single, tertiary care, urban children's hospital. Patients presenting directly to our emergency department aged 2 to 18 years were included if they had a diagnosis of severe asthma exacerbation, defined by an initial Respiratory Clinical Score (RCS) of 9 or higher. A total of 787 patients were identified during the study timeframe (December 16, 2017, to December 31, 2018), and of those, 651 patients met study criteria and were included in the analysis. The χ2 test was used to establish P values for categorical variables. For normally distributed variables, a t test was used. For nonnormally distributed variables, the Kruskal-Wallis test was used. A P value of 0.05 or less was interpreted as statistically significant. RESULTS: Patients who received terbutaline had an increased risk of admission to the PICU (P < 0.001). This association was lost after controlling for age, sex, continuous albuterol use, and intramuscular epinephrine use (P = 0.362). Patients receiving terbutaline in the emergency department also had a higher risk of admission to the hospital (odds ratio, 1.55; confidence interval, 1.08-2.22; P = 0.020) as compared with their nonterbutaline counterparts. Overall, patients in the terbutaline group had a higher initial RCS at presentation. Upon further analysis, patients with the same RCS at presentation were more likely to be admitted if they received terbutaline than those who did not. There was no statistically significant difference in length of stay (P = 0.298) and BiPAP/CPAP use (P = 0.107). The patients on terbutaline were relatively more likely to require oxygen (P = 0.003) and intramuscular epinephrine (P = 0.010) than the patients not on terbutaline. CONCLUSIONS: Terbutaline administration given to pediatric patients experiencing a severe asthma exacerbation was not associated with decreased PICU or general hospital floor admission. The study is limited given that it was a retrospective analysis. Further randomized controlled trials are needed to assess the role of terbutaline in severe acute asthma exacerbations in pediatric patients.

Two cryptosporidia species encode active creatine kinases that are not seen in other apicomplexa species.

A gene encoding creatine kinase was identified in two cryptosporidia species, Cryptosporidium muris and C. andersonii. They were syntenic and shared 91% identity 94% identity at the amino acid level and nucleotide levels respectively. The C. muris creatine kinase was characterized biochemically and shown to phosphorylate both creatine and glycocyamine with a 20-fold greater preference for creatine. The observed catalytic turnover with creatine was kcat = 30 s-1 with a catalytic efficiency of 15.4 mM-1 s-1. These values were within the range observed for other creatine kinases. A search of all the apicomplexa genomes available on EuPathDB did not reveal any other phosphagen kinase genes raising the possibility of horizontal gene transfer. However, no definitive conclusion could be drawn regarding this hypothesis given the massive amount of gene loss in the apicomplexa species which are primarily parasitic species. The implications of a creatine kinase in the parasites' infection cycle are discussed.

Bacterial arginine kinases have a highly skewed distribution within the proteobacteria.

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. A recent search of the available bacterial genomes revealed 49 unique sequences that appear to code for an arginine kinase (AK). The distribution of sequences was highly skewed with thirty nine out the forty nine sequences being found in six Proteobacteria classes (α, ß, δ, γ, ε, and ζ) which represented 46.6% of the 61,335 bacterial genomes available at JGI-IMG/M website. Moreover, twenty one of the unique and metagenome bAK sequences identified were from δ-Proteobacteria despite these representing only 0.88% of the total genomes available. Phylogenetic analyses revealed that the bacterial AK sequences were interpersed between basal species such as cnidarians, sponges and protozoa, displaying an unstable clustering that was dependent upon the parameters chosen for phylogenetic analysis. Three of these putative bacterial AK genes were cloned into the pET45 expression vector, expressed, and biochemically confirmed to be capable of phosphorylating arginine using ATP. Results of the kinetic analyses of the putative bAKs from Ahrensia, D. autotrophicum, and O. profundus show that the catalytic efficiencies with respect to arginine for each enzyme, measured at 104-105 M-1 s-1, fall within the range expected for competent arginine kinases.

Mechanism of 6-Hydroxynicotinate 3-Monooxygenase, a Flavin-Dependent Decarboxylative Hydroxylase Involved in Bacterial Nicotinic Acid Degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.

A Randomized Trial Comparing Metered Dose Inhalers and Breath Actuated Nebulizers.

BACKGROUND: Despite little evidence for its effectiveness, the breath-actuated nebulizer (BAN) is the default albuterol delivery method in our pediatric emergency department. OBJECTIVE: We compared the clinical efficacy of BAN and the metered-dose inhaler (MDI) in treating subjects patients 2 to 17 years of age who presented with mild to moderate asthma exacerbations. METHODS: This is a randomized, nonblinded, noninferiority study conducted at a single pediatric tertiary care emergency department. Subjects presenting with a Pediatric Asthma Score ranging from 5 to 11 received albuterol by BAN or MDI via standard weight-based and symptom severity dosing protocols. Aerosolized ipratropium (via BAN) and intravenous magnesium sulfate were given when clinically indicated. The primary outcome was patient disposition. The noninferiority margin for the primary outcome was an admission rate difference ≤10%. Analyses were adjusted for confounders that were significant at p ≤ 0.10. RESULTS: We enrolled 890 subjects between October 2014 and April 2015. BAN and MDI groups were comparable for age, gender, and race but not for pretreatment symptom severity; 51% in the MDI group had a Pediatric Asthma Score of moderate severity (8-11) vs. 63% in the BAN group (p < 0.003). Unadjusted admission rates were 11.9% for MDI and 12.8% for BAN, for an unadjusted risk difference of -0.9% (95% confidence interval -5% to 3%). After adjusting for baseline confounder severity, the risk difference was 2% (95% confidence interval -4% to 7%), which met the criteria for noninferiority. CONCLUSIONS: Albuterol therapy by MDI is noninferior to BAN for the treatment of mild to moderate asthma exacerbations in children 2 to 17 years of age.

Characterization of the arginine kinase isoforms in Caenorhabditis elegans.

Phosphagen kinases (PKs) are well-studied enzymes involved in energy homeostasis in a wide range of animal, protozoan, and even some bacterial species. Recent genome efforts have allowed comparative work on the PKs to extend beyond the biochemistry of individual proteins to the comparative cellular physiology and examining of the role of all PK family members in an organism. The sequencing of the Caenorhabditis elegans genome and availability of sophisticated genetic tools within that system affords the opportunity to conduct a detailed physiological analysis of the PKs from a well known invertebrate for comparison with the extensive work conducted on vertebrate systems. As a first step in this effort we have carried out a detailed molecular genetic and biochemical characterization of the PKs in C. elegans. Our results reveal that C. elegans has five PK genes encoding arginine kinases that range in catalytic efficiency (kcat/KM(Arg)) from (3.1±0.6)×10(4) to (9±4)×10(5) M(-1) s(-1). This range is generally within the range seen for arginine kinases from a variety of species. Our molecular genetic and phylogenetic analysis reveals that the gene family has undergone extensive intron loss and gain within the suborder Rhabditina. In addition, within C. elegans we find evidence of gene duplication and loss. The analysis described here for the C. elegans AKs represents one of the most complete biochemical and molecular genetic analysis of a PK family within a genetically tractable invertebrate system and opens up the possibility of conducting detailed physiological comparisons with vertebrate systems using the sophisticated tools available with this model invertebrate system.

Draft Genome Sequence of the Nicotinate-Metabolizing Soil Bacterium Bacillus niacini DSM 2923.

Bacillus niacini is a member of a small yet diverse group of bacteria able to catabolize nicotinic acid. We report here the availability of a draft genome for B. niacini, which we will use to understand the evolution of its namesake phenotype, which appears to be unique among the species in its phylogenetic neighborhood.

Characterization of a putative oomycete taurocyamine kinase: Implications for the evolution of the phosphagen kinase family.

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. Within animal species, these enzymes play a critical role in energy homeostasis by catalyzing the reversible transfer of a high-energy phosphoryl group from Mg⋅ATP to an acceptor molecule containing a guanidinium group. In this work, a putative PK gene was identified in the oomycete Phytophthora sojae that was predicted, based on sequence homology, to encode a multimeric hypotaurocyamine kinase. The recombinant P. sojae enzyme was purified and shown to catalyze taurocyamine phosphorylation efficiently (kcat/KM (taurocyamine) = 2 × 10(5) M(-1) s(-1)) and glycocyamine phosphorylation only weakly (kcat/KM (glycocyamine) = 2 × 10(2) M(-1) s(-1)), but lacked any observable kinase activity with the more ubiquitous guanidinium substrates, creatine or arginine. Additionally, the enzyme was observed to be dimeric but lacked cooperativity between the subunits in forming a transition state analog complex. These results suggest that protozoan PKs may exhibit more diversity in substrate specificity than was previously thought.

Flight of a cytidine deaminase complex with an imperfect transition state analogue inhibitor: mass spectrometric evidence for the presence of a trapped water molecule.

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (K(d) ~ 10(-12) M), capturing all but ~5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (K(tx) ~ 10(-16) M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme-inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364-11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E(2)Zn(2)W(2) (dimeric Zn-CDA with two water molecules), E(2)Zn(2)W(2)Fz, and E(2)Zn(2)W(2)Fz(2), where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E(2)Zn(2) is the only dimeric species detected, with no additional water molecules. Experiments conducted in H(2)(18)O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E(2)Zn(2)) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (k(off) > 0.14 s(-1)) much more rapidly than is 5-fluorozebularine (k(off) = 0.014 s(-1)), despite the greater thermodynamic stability of the zebularine complex.

Identification and characterization of a putative arginine kinase homolog from Myxococcus xanthus required for fruiting body formation and cell differentiation.

Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.

Structure and catalytic mechanism of nicotinate (vitamin B3) degradative enzyme maleamate amidohydrolase from Bordetella bronchiseptica RB50.

The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 µM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.

Characterization of a novel bacterial arginine kinase from Desulfotalea psychrophila.

Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp. PE36 have revealed what appears to be a phosphagen kinase homolog present in their genomes. Based on sequence comparisons these putative homologs bear a strong resemblance to arginine kinases found in many invertebrates and some protozoa. We describe here a biochemical characterization of one of these homologs from D. psychrophila expressed in E. coli that confirms its ability to reversibly catalyze phosphoryl transfer from ATP to arginine. A phylogenetic analysis suggests that these bacteria homologs are not widely distributed in proteobacteria species. They appear more related to protozoan arginine kinases than to similar proteins seen in some Gram-positive bacteria that share key catalytic residues but encode protein tyrosine kinases. This raises the possibility of horizontal gene transfer as a likely origin of the bacterial arginine kinases.

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site.

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.