The aspartic residue at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through sitedirected mutagenesis. The wild-type (with in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinylAla-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methylcoumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp'l8trypsin, the activity of [Ser'"1trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2-to 6-fold. In contrast, [Serl89]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser"]9]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: El, > EPP > EPA > EIP EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.The binding of substrates or inhibitors to the specificity pocket of an enzyme involves a combination of chemical forces including hydrogen bonds and electrostatic, hydrophobic, and steric interactions. The complexity of the interactions involved in the substrate specificity of an enzyme is exemplified by trypsin. The three-dimensional structures of trypsin bound to pancreatic trypsin inhibitor (PTI) (1)(2)(3)(4) or to the pseudosubstrate benzamidine (5, 6) suggest that the carboxylate of Asp-189, at the base of the trypsin binding pocket, is largely responsible for the specificity of binding of the enzyme to positively charged amino acid side chains.The major role of electrostatic interactions in the trypsin binding pocket has been analyzed by measuring (7) and calculating (8) the stabilization energies of binding between a series of benzamidine analogs and trypsin. In addition, the high degree of structural similarity of the trypsin and chymotrypsin binding pockets (9, 10) is consistent with the experimental observations that aromatic side chains may form favorable hydrophobic interactions with the trypsin binding pocket (10-13 by using a series of synthetic fluorogenic substrates with various amino acids in the C-terminal (P1) position in order to compare the electrostatic interactions of the different enzyme-substrate pairs. MATERIALS AND METHODSMaterials. Tetrapeptide substrates with the fluorogenic leaving...

Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory ␥2 subunit have been shown to cause an essentially cardiacrestricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart.Objective: The ␥ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of ␥2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results:A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of ␥2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca 2؉ Key Words: familial hypertrophic cardiomyopathy Ⅲ myocardial contractility Ⅲ phosphorylation A MP-activated protein kinase (AMPK) is a crucial component of a highly conserved serine/threonine protein kinase cascade central to the control of energy balance at the cellular and whole-body levels. 1,2 AMPK exists as a ␣␤␥ heterotrimer, with ␣ being the catalytic subunit, and the ␤ and ␥ subunits performing structural and regulatory functions. Isoforms of all subunits have been identified (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3), each being encoded by a different gene (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, and PRKAG3, respectively). The ␣ subunits consist of a typical serine/threonine protein kinase domain at the N-terminus (which also contains the critical phosphorylation site for AMPK activation, Thr172 3 ) and a C-terminal domain involved in the binding of the ␤ and ␥ subunits. 1,2 The ␤ subunits are myristoylated at their N-terminus, contain a conserved C-terminal domain that is involved in binding of the ␣ and ␥ subunits, and a carbohydrate binding domain. The carbohydrate binding domain may allow AMPK to sense the status of cellular energy reserves in the form of glycogen in addition to responding to AMP/ATP levels. 4 The ␥ subunits have a high degree of homology in their C-terminal Original received October 31, 2011; revision received March 14, 2012; accepted March 19, 2012. In February 2012 sequences, all containing 2 pairs of highly conserved cystathionine ␤-synthase domains, which have been shown to be directly involved in the binding of adenine nucleotides. [5][6][7] In contrast, their N-terminal regions are highly variable, with ␥2 and ␥3 possessing different long N-terminal extensions compared with the shorter ␥1 isoform (Figure 1). The ␥2 and ␥3 N-terminal sequences appear to be unique in that they do not share sequence identity with each other nor with any known protein. ...

SummaryDespite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Arylamine N-acetyltransferases (NATs) are polymorphic xenobiotic metabolising enzymes, linked to cancer susceptibility in a variety of tissues. In humans and in mice there are multiple NAT isoforms. To identify whether the different isoforms represent inbuilt redundancy or whether they have unique roles, we have generated mice with a null allele of Nat2 by gene targeting. This mouse line conclusively demonstrates that the different isoforms have distinct functions with no compensatory expression in the Nat2 null animals of the other isoforms. In addition, we have used the transgenic line to show the pattern of Nat2 expression during development. Although Nat2 is not essential for embryonic development, it has a widespread tissue distribution from at least embryonic day 9.5. This mouse line now paves the way for the teratological role of Nat2 to be tested.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. The NATs have been shown to be important in drug detoxification and carcinogen activation, with humans possessing two isoenzymes encoded by polymorphic genes. This polymorphism has pharmacogenetic implications, leading to different rates of inactivation of drugs, including the anti-tubercular agent isoniazid and the anti-hypertensive drug hydralazine. Mice provide a good model for human NAT, allowing genetic manipulation of expression to explore possible endogenous roles of these enzymes. The first three-dimensional NAT structure was resolved for NAT from Salmonella typhimurium, and subsequently the structure of NAT from Mycobacterium smegmatis has been elucidated. These identified a 'Cys-His-Asp' catalytic triad (conserved in all NATs), which is believed to be responsible for the activation of the active site cysteine residue. As more genomic data become available, NAT homologues continue to be found in prokaryotic species, many of which are pathogenic, including Mycobacterium tuberculosis. The discovery of NAT in M. tuberculosis is particularly significant, since this enzyme participates in inactivation of isoniazid in the bacterium, with implications for isoniazid resistance. Structural studies on NAT proteins and phenotypic analyses of organisms (both mice and prokaryotes) following genetic modifications of the nat genes are leading to an understanding of the potentially diverse roles of NAT in endogenous and xenobiotic metabolism. These studies have indicated that NAT, particularly in Mycobacteria, has the potential to be a drug target. Combinatorial chemical approaches, together with in silico structural studies, will allow for advances in the identification of NAT substrates and inhibitors, both as experimental tools and as potential drugs.

The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca2+ channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca2+ influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

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